Oligonucleotide compositions and methods for the modulation of the expression of B7 protein

ABSTRACT

Compositions and methods for the treatment of asthma with oligonucleotides which specifically hybridize with nucleic acids encoding B7 proteins.

RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.10/641,962, filed Aug. 15, 2003, now allowed, which is a applicationclaiming the benefit under 35 USC 119(e) of U.S. Provisional PatentApplication Ser. No. 60/651,504, filed May 23, 2003, now expired; eachof which is herein incorporated by reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0020US.C1, created on May 14, 2007 which is 180 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to diagnostics, research reagents andtherapeutics for disease states which respond to modulation of T cellactivation. In particular, this invention relates to antisenseoligonucleotide interactions with certain messenger ribonucleic acids(mRNAs) or DNAs involved in the synthesis of proteins that modulate Tcell activation. Antisense oligonucleotides designed to hybridize tonucleic acids encoding B7 proteins are provided. These oligonucleotideshave been found to lead to the modulation of the activity of the RNA orDNA, and thus to the modulation of T cell activation. Palliative,therapeutic and prophylactic effects result.

BACKGROUND OF THE INVENTION

Inflammation is a localized protective response mounted by tissues inresponse to injury, infection, or tissue destruction resulting in thedestruction of the infectious or injurious agent and isolation of theinjured tissue. A typical inflammatory response proceeds as follows:recognition of an antigen as foreign or recognition of tissue damage,synthesis and release of soluble inflammatory mediators, recruitment ofinflammatory cells to the site of infection or tissue damage,destruction and removal of the invading organism or damaged tissue, anddeactivation of the system once the invading organism or damage has beenresolved. In many human diseases with an inflammatory component, thenormal, homeostatic mechanisms which attenuate the inflammatoryresponses are defective, resulting in damage and destruction of normaltissue.

Cell-cell interactions are involved in the activation of the immuneresponse at each of the stages described above. One of the earliestdetectable events in a normal inflammatory response is adhesion ofleukocytes to the vascular endothelium, followed by migration ofleukocytes out of the vasculature to the site of infection or injury. Ingeneral, the first inflammatory cells to appear at the site ofinflammation are neutrophils, followed by monocytes and lymphocytes.Cell-cell interactions are also critical for activation of bothB-lymphocytes (B cells) and T-lymphocytes (T cells) with resultingenhanced humoral and cellular immune responses, respectively.

The hallmark of the immune system is its ability to distinguish betweenself (host) and nonself (foreign invaders). This remarkable specificityexhibited by the immune system is mediated primarily by T cells. T cellsparticipate in the host's defense against infection but also mediateorgan damage of transplanted tissues and contribute to cell attack ingraft-versus-host disease (GVHD) and some autoimmune diseases. In orderto induce an antigen-specific immune response, a T cell must receivesignals delivered by an antigen-presenting cell (APC). T cell-APCinteractions can be divided into three stages: cellular adhesion, T cellreceptor (TCR) recognition, and costimulation. At least two discretesignals are required from an APC for induction of T cell activation. Thefirst signal is antigen-specific and is provided when the TCR interactswith an antigen in the context of a major histocompatibility complex(MHC) protein, or an MHC-related CD1 protein, expressed on the surfaceof an APC (“CD,” standing for “cluster of differentiation,” is a termused to denote different T cell surface molecules). The second(costimulatory) signal involves the interaction of the T cell surfaceantigen, CD28, with its ligand on the APC, which is a member of the B7family of proteins.

CD28, a disulfide-linked homodimer of a 44 kilodalton polypeptide and amember of the immunoglobulin superfamily, is one of the majorcostimulatory signal receptors on the surface of a resting T cell for Tcell activation and cytokine production (Allison, Curr. Opin. Immunol.,1994, 6, 414; Linsley and Ledbetter, Annu. Rev. Immunol., 1993, 11, 191;June et al., Immunol. Today, 1994, 15, 321). Signal transduction throughCD28 acts synergistically with TCR signal transduction to augment bothinterleukin-2 (IL-2) production and proliferation of naive T cells. B7-1(also known as CD80) was the first ligand identified for CD28 (Liu andLinsley, Curr. Opin. Immunol., 1992, 4, 265). B7-1 is normally expressedat low levels on APCs, however, it is upregulated following activationby cytokines or ligation of cell surface molecules such as CD40(Lenschow et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 11054; Nabaviet al., Nature, 1992, 360, 266). Initial studies suggested that B7-1 wasthe CD28 ligand that mediated costimulation (Reiser et al., Proc. Natl.Acad. Sci. U.S.A., 1992, 89, 271; Wu et al., J. Exp. Med., 1993, 178,1789; Harlan et al., Proc. Natl. Acad. Sci. U.S.A., 1994, 91, 3137).However, the subsequent demonstration that anti-B7-1 monoclonalantibodies (mAbs) had minimal effects on primary mixed lymphocytereactions and that B7-1-deficient mice responded normally to antigens(Lenschow et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 11054;Freeman et al., Science, 1993, 262, 909) resulted in the discovery of asecond ligand for the CD28 receptor, B7-2 (also known as CD86). Incontrast with anti-B7-1 mAbs, anti-B7-2 mAbs are potent inhibitors of Tcell proliferation and cytokine production (Wu et al., J. Exp. Med.,1993, 178, 1789; Chen et al., J. Immunol., 1994, 152, 2105; Lenschow etal., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 11054). B7:CD28 signalingmay be a necessary component of other T cell costimulatory pathways,such as CD40:CD40L (CD40 ligand) signaling (Yang et al., Science, 1996,273, 1862).

In addition to binding CD28, B7-1 and B7-2 bind the cytolyticT-lymphocyte associated protein CTLA4. CTLA4 is a protein that isstructurally related to CD28 but is expressed on T cells only afteractivation (Linsley et al., J. Exp. Med., 1991, 174, 561). A solublerecombinant form of CTLA4, CTLA4-Ig, has been determined to be a moreefficient inhibitor of the B7:CD28 interaction than monoclonalantibodies directed against CD28 or a B7 protein. In vivo treatment withCTLA4-Ig results in the inhibition of antibody formation to sheep redblood cells or soluble antigen (Linsley et al., Science, 1992, 257,792), prolongation of cardiac allograft and pancreatic islet xenograftsurvival (Lin et al., J. Exp. Med., 1993, 178, 1801; Lenschow et al.,1992, Science, 257, 789; Lenschow et al., Curr. Opin. Immunol., 1991, 9,243), and significant suppression of immune responses in GVHD (Hakim etal., J. Immun., 1995, 155, 1760). It has been proposed that CD28 andCTLA4, although both acting through common B7 receptors, serve opposingcostimulatory and inhibitory functions, respectively (Allison et al.,Science, 1995, 270, 932). CTLA4-Ig, which binds both B7-1 and B7-2molecules on antigen-presenting cells, has been shown to block T-cellcostimulation in patients with stable psoriasis vulgaris, and to cause a50% or greater sustained improvement in clinical disease activity in 46%of the patients to which it was administered. This result wasdose-dependent. Abrams et al., J. Clin. Invest., 1999, 9, 1243-1225.

European Patent Application No. EP 0 600 591 discloses a method ofinhibiting tumor cell growth in which tumor cells from a patient arerecombinantly engineered ex vivo to express a B7-1 protein and thenreintroduced into a patient. As a result, an immunologic response isstimulated against both B7-transfected and nontransfected tumor cells.

International Publication No. WO95/03408 discloses nucleic acidsencoding novel CTLA4/CD28 ligands which costimulate T cell activation,including B7-2 proteins. Also disclosed are antibodies to B7-2 proteinsand methods of producing B7-2 proteins.

International Publication No. WO95/05464 discloses a polypeptide, otherthan B7-1, that binds to CTLA4, CD28 or CTLA4-Ig. Also disclosed aremethods for obtaining a nucleic acid encoding such a polypeptide.

International Publication No. WO 95/06738 discloses nucleic acidsencoding B7-2 (also known as B70) proteins. Also disclosed areantibodies to B7-2 proteins and methods of producing B7-2 proteins.

European Patent Application No. EP 0 643 077 discloses a monoclonalantibody which specifically binds a B7-2 (also known as B70) protein.Also disclosed are methods of producing monoclonal antibodies whichspecifically bind a B7-2 protein.

U.S. Pat. No. 5,434,131 discloses the CTLA4 protein as a ligand for B7proteins. Also disclosed are methods of producing CTLA4 fusion proteins(e.g., CTLA4-Ig) and methods of regulating immune responses usingantibodies to B7 proteins or CTLA4 proteins.

International Publication No. WO95/22619 discloses antibodies specificto B7-1 proteins which do not bind to B7-2 proteins. Also disclosed aremethods of regulating immune responses using antibodies to B7-1proteins.

International Publication No. WO95/34320 discloses methods forinhibiting T cell responses using a first agent which inhibits acostimulatory agent, such as a CTLA4-Ig fusion protein, and a secondagent which inhibits cellular adhesion, such as an anti-LFA-1 antibody.Such methods are indicated to be particularly useful for inhibiting therejection of transplanted tissues or organs.

International Publication No. WO95/32734 discloses Fc RII bridgingagents which either prevent the upregulation of B7 molecules or impairthe expression of ICAM-3 on antigen presenting cells. Such FcRIIbridging agents include proteins such as aggregated human IgG moleculesor aggregated Fc fragments of human IgG molecules.

International Publication No. WO96/11279 discloses recombinant virusescomprising genetic sequences encoding (1) one or more immunostimulatoryagents, including B7-1 and B7-2, and (2) antigens from a disease causingagent. Also disclosed are methods of treating diseases using suchrecombinant viruses.

To date, there are no known therapeutic agents which effectivelyregulate and prevent the expression of B7 proteins such as B7-1 andB7-2. Thus, there is a long-felt need for compounds and methods whicheffectively modulate critical costimulatory molecules such as the B7proteins.

SUMMARY OF THE INVENTION

In accordance with the present invention, oligonucleotides are providedwhich specifically hybridize with nucleic acids encoding B7-1 or B7-2.Certain oligonucleotides of the invention are designed to bind eitherdirectly to mRNA transcribed from, or to a selected DNA portion of, theB7-1 or B7-2 gene, thereby modulating the amount of protein translatedfrom a B7-1 or B7-2 mRNA or the amount of mRNA transcribed from a B7-1or B 7-2 gene, respectively.

Oligonucleotides may comprise nucleotide sequences sufficient inidentity and number to effect specific hybridization with a particularnucleic acid. Such oligonucleotides are commonly described as“antisense.” Antisense oligonucleotides are commonly used as researchreagents, diagnostic aids, and therapeutic agents.

It has been discovered that the B7-1 and B7-2 genes, encoding B7-1 andB7-2 proteins, respectively, are particularly amenable to this approach.As a consequence of the association between B7 expression and T cellactivation and proliferation, inhibition of the expression of B7-1 orB7-2 leads to inhibition of the synthesis of B7-1 or B7-2, respectively,and thereby inhibition of T cell activation and proliferation.Additionally, the oligonucleotides of the invention may be used toinhibit the expression of one of several alternatively spliced mRNAs ofa B7 transcript, resulting in the enhanced expression of otheralternatively spliced B7 mRNAs. Such modulation is desirable fortreating various inflammatory or autoimmune disorders or diseases, ordisorders or diseases with an inflammatory component such as asthma,juvenile diabetes mellitus, myasthenia gravis, Graves' disease,rheumatoid arthritis, allograft rejection, inflammatory bowel disease,multiple sclerosis, psoriasis, lupus erythematosus, systemic lupuserythematosus, diabetes, multiple sclerosis, contact dermatitis,rhinitis, various allergies, and cancers and their metastases. Suchmodulation is further desirable for preventing or modulating thedevelopment of such diseases or disorders in an animal suspected ofbeing, or known to be, prone to such diseases or disorders.

In one embodiment, the invention provides methods of inhibiting theexpression of a nucleic acid molecule encoding B7-1 or B7-2 in anindividual, comprising the step of administering to said individual acompound of the invention targeted to a nucleic acid molecule encodingB7-1 or B7-2, wherein said compound specifically hybridizes with andinhibits the expression of a nucleic acid molecule encoding B7-1 orB7-2.

The invention further provides methods of inhibiting expression of anucleic acid molecule encoding B7-1 or B7-2 in an individual, comprisingthe step of administering to an individual a compound of the inventionwhich specifically hybridizes with at least an 8-nucleobase portion ofan active site on a nucleic acid molecule encoding B7-1 or B7-2. Regionsin the nucleic acid which when hybridized to a compound of the inventioneffect significantly lower B7-1 or B7-2 expression compared to acontrol, are referred to as active sites.

The invention also provides methods of inhibiting expression of anucleic acid molecule encoding B7-1 or B7-2 in an individual, comprisingthe step of administering a compound of the invention targeted to anucleic acid molecule encoding B7-1 or B7-2, wherein the compoundspecifically hybridizes with the nucleic acid and inhibits expression ofB7-1 or B7-2.

In another aspect the invention provides methods of inhibitingexpression of a nucleic acid molecule encoding B7-1 or B7-2 in anindividual, comprising the step of administering a compound of theinvention targeted to a nucleic acid molecule encoding B7-1 or B7-2,wherein the compound specifically hybridizes with the nucleic acid andinhibits expression of B7-1 or B7-2, said compound comprising at least 8contiguous nucleobases of any one of the compounds of the invention.

The invention also provides methods of inhibiting the expression of anucleic acid molecule encoding B7-1 or B7-2 in an individual, comprisingthe step of administering a compound of the invention targeted to anucleic acid molecule encoding B7-1 or B7-2, wherein the compoundspecifically hybridizes with an active site in the nucleic acid andinhibits expression of B7-1 or B7-2, and the compound comprises at least8 contiguous nucleobases of any one of the compounds of the invention.

In another aspect the invention provides methods of inhibitingexpression of a nucleic acid molecule encoding B7-1 or B7-2 in anindividual, comprising the step of administering an oligonucleotidemimetic compound targeted to a nucleic acid molecule encoding B7-1 orB7-2, wherein the compound specifically hybridizes with the nucleic acidand inhibits expression of B7-1 or B7-2, and the compound comprises atleast 8 contiguous nucleobases of a compound of the invention.

In another aspect, the invention provides methods of inhibiting theexpression of a nucleic acid molecule encoding B7-1 or B7-2 in anindividual comprising the step of administering a compound of theinvention targeted target to a nucleic acid encoding B7-1 or B7-2,wherein the compound inhibits B7-1 or B7-2 mRNA expression by at least5% in 80% confluent HepG2 cells in culture at an optimum concentrationcompared to a control. In yet another aspect, the compounds inhibitexpression of mRNA encoding B7-1 or B7-2 in 80% confluent HepG2 cells inculture at an optimum concentration by at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, or atleast 50%, compared to a control.

The invention also relates to pharmaceutical compositions which comprisean antisense oligonucleotide to a B7 protein in combination with asecond anti-inflammatory agent, such as a second antisenseoligonucleotide to a protein which mediates intercellular interactions,e.g., an intercellular adhesion molecule (ICAM) protein.

Methods comprising contacting animals with oligonucleotides specificallyhybridizable with nucleic acids encoding B7 proteins are hereinprovided. These methods are useful as tools, for example, in thedetection and determination of the role of B7 protein expression invarious cell functions and physiological processes and conditions, andfor the diagnosis of conditions associated with such expression. Suchmethods can be used to detect the expression of B7 genes (i.e., B7-1 orB7-2) and are thus believed to be useful both therapeutically anddiagnostically. Methods of modulating the expression of B7 proteinscomprising contacting animals with oligonucleotides specificallyhybridizable with a B7 gene are herein provided. These methods arebelieved to be useful both therapeutically and diagnostically as aconsequence of the association between B7 expression and T cellactivation and proliferation. The present invention also comprisesmethods of inhibiting B7-associated activation of T cells using theoligonucleotides of the invention. Methods of treating conditions inwhich abnormal or excessive T cell activation and proliferation occursare also provided. These methods employ the oligonucleotides of theinvention and are believed to be useful both therapeutically and asclinical research and diagnostic tools. The oligonucleotides of thepresent invention may also be used for research purposes. Thus, thespecific hybridization exhibited by the oligonucleotides of the presentinvention may be used for assays, purifications, cellular productpreparations and in other methodologies which may be appreciated bypersons of ordinary skill in the art.

The methods disclosed herein are also useful, for example, as clinicalresearch tools in the detection and determination of the role of B7-1 orB7-2 expression in various immune system functions and physiologicalprocesses and conditions, and for the diagnosis of conditions associatedwith their expression. The specific hybridization exhibited by theoligonucleotides of the present invention may be used for assays,purifications, cellular product preparations and in other methodologieswhich may be appreciated by persons of ordinary skill in the art. Forexample, because the oligonucleotides of this invention specificallyhybridize to nucleic acids encoding B7 proteins, sandwich and otherassays can easily be constructed to exploit this fact. Detection ofspecific hybridization of an oligonucleotide of the invention with anucleic acid encoding a B7 protein present in a sample can routinely beaccomplished. Such detection may include detectably labeling anoligonucleotide of the invention by enzyme conjugation, radiolabeling orany other suitable detection system. A number of assays may beformulated employing the present invention, which assays will commonlycomprise contacting a tissue or cell sample with a detectably labeledoligonucleotide of the present invention under conditions selected topermit hybridization and measuring such hybridization by detection ofthe label, as is appreciated by those of ordinary skill in the art.

The present invention provides an antisense oligonucleotide whichspecifically hybridizes to a nucleic acid encoding human B7.2 protein,said antisense oligonucleotide comprising at least an 8 nucleobaseportion of SEQ ID NO: 374, 391 or 440, wherein said antisenseoligonucleotide inhibits expression of said human B7.2 protein.

In one aspect, the invention provides the antisense oligonucleotide ofthe invention, wherein said antisense oligonucleotide has the sequenceshown in SEQ ID NO: 374, 391 or 440.

In another aspect, the antisense oligonucleotide of the invention has atleast one modified internucleotide linkage.

In yet another aspect, the invention encompasses the antisenseoligonucleotide of the invention wherein said modified linkage is aphosphorothioate. The antisense oligonucleotide of claim 2, wherein allinternucleotide linkages are phosphorothioate linkages.

In another aspect, the invention encompasses the antisenseoligonucleotide of the invention having at least one 2′ sugarmodification. The antisense oligonucleotide of claim 2, whereinnucleotides 1-5 and 16-20 comprise 2′-MOE modifications.

In yet another aspect, the invention provides the antisenseoligonucleotide of the invention wherein said 2′ sugar modification is a2′-MOE.

In another aspect, the invention encompasses the antisenseoligonucleotide of the invention having at least one base modification.

In another aspect, the invention provides the antisense oligonucleotideof the invention wherein said base modification is a 5-methylcytidine.The antisense oligonucleotide of claim 2, wherein all cytidine residuesare replaced with 5′methylcytidines.

In yet another aspect, the invention provides an antisenseoligonucleotide having the sequence of SEQ ID NO: 374, 391 or 440,wherein all internucleotide linkages are phosphorothioate linkages, allcytidine residues are replaced with 5′methylcytidines and nucleotides1-15 and 16-20 comprise 2′-MOE modifications.

In another aspect, the invention also provides a method of inhibitingexpression of human B7.2 protein in cells or tissues comprisingcontacting said cells or tissues with the antisense oligonucleotide ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the inhibitory effect of the indicatedoligonucleotides on B7-1 protein expression in COS-7 cells.

FIG. 2 is a dose-response curve showing the inhibitory effect ofoligonucleotides on cell surface expression of B7-1 protein. Solid line,ISIS 13812; dashed line, ISIS 13800; dotted line, ISIS 13805.

FIG. 3 is a bar graph showing the inhibitory effect of the indicatedoligonucleotides on cell surface expression of B7-2 in COS-7 cells.

FIG. 4 is a bar graph showing the inhibitory effect of the indicatedoligonucleotides, including ISIS 10373 (a 20-mer) and ISIS 10996 (a15-mer) on cell surface expression of B7-2 in COS-7 cells.

FIG. 5 is a bar graph showing the specificity of inhibition of B7-1 orB7-2 protein expression by oligonucleotides. Cross-hatched bars, B7-1levels; striped bars, B7-2 levels.

FIG. 6 is a dose-response curve showing the inhibitory effect ofoligonucleotides having antisense sequences to ICAM-1 (ISIS 2302) orB7-2 (ISIS 10373) on cell surface expression of the ICAM-1 and B7-2proteins. Solid line with X's, levels of B7-1 protein on cells treatedwith ISIS 10373; dashed line with asterisks, levels of ICAM-1 protein oncells treated with ISIS 10373; solid line with triangles, levels of B7-1protein on cells treated with ISIS 2302; solid line with squares, levelsof ICAM-1 protein on cells treated with ISIS 10373.

FIG. 7 is a bar graph showing the effect of the indicatedoligonucleotides on T cell proliferation.

FIG. 8 is a dose-response curve showing the inhibitory effect ofoligonucleotides on murine B7-2 protein expression in COS-7 cells. Solidline with asterisks, ISIS 11696; dashed line with triangles, ISIS 11866.

FIG. 9 is a bar graph showing the effect of oligonucleotides ISIS 11696and ISIS 11866 on cell surface expression of murine B7-2 protein inIC-21 cells. Left (black) bars, no oligonucleotide; middle bars, 3 μMindicated oligonucleotide; right bars, 10 μM indicated oligonucleotide.

FIG. 10 is a graph showing the effect of ISIS 17456 on severity of EAEat various doses.

FIG. 11A-B are graphs showing the detection of B7.2 mRNA (FIG. 11A) andB7.1 mRNA (FIG. 11B) during the development of ovalbumin-induced asthmain a mouse model.

FIG. 12 is a graph showing that intratracheal administration of ISIS121874, an antisense oligonucleotide targeted to mouse B7.2, followingallergen challenge, reduces the airway response to methacholine.

FIG. 13 is a graph showing the dose-dependent inhibition of the Penhresponse to 50 mg/ml methacholine by ISIS 121874. Penh is adimensionless parameter that is a function of total pulmonary airflow inmice (i.e., the sum of the airflow in the upper and lower respiratorytracts) during the respiratory cycle of the animal. The lower the Penh,the greater the airflow. The dose of ISIS 121874 is shown on the x-axis.

FIG. 14 is a graph showing the inhibition of allergen-inducedeosinophilia by ISIS 121874. The dose of ISIS 121874 is shown on thex-axis.

FIG. 15 is a graph showing the lung concentration-dose relationship forISIS 121874 delivered by intratracheal administration.

FIG. 16 is a graph showing the retention of ISIS 121874 in lung tissuefollowing single dose (0.3 mg/kg) intratracheal instillation in theovalbumin-induced mouse model of asthma.

FIG. 17 is a graph showing the effects of ISIS 121874, a 7 base pairmismatched control oligonucleotide (ISIS 131906) and a gap ablatedcontrol oligonucleotide which does not promote cleavage by RNase H (ISIS306058).

FIGS. 18A-B are graphs showing the effect of ISIS 121874 on B7.2 (FIG.18A) and B7.1 (FIG. 18B) mRNA in lung tissue of ovalbumin-challengedmice.

FIGS. 19A-B are graphs showing the effect of ISIS 121874 on B7.2 (FIG.19A) and B7.1 (FIG. 19B) mRNA in draining lymph nodes ofovalbumin-challenged mice.

FIG. 20 is a graph showing that treatment with an antisenseoligonucleotide targeted to B7.1 (ISIS 121844) reduces allergen-inducedeosinophilia in the ovalbumin-induced mouse model of asthma.

FIGS. 21A-B are graphs showing that treatment with ISIS 121844 reducesthe levels of B7.1 mRNA (FIG. 21A) and B7.2 mRNA (FIG. 21B) in the mouselung.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligonucleotides for use in antisenseinhibition of the function of RNA and DNA encoding B7 proteins includingB7-1 and B7-2. The present invention also employs oligonucleotides whichare designed to be specifically hybridizable to DNA or messenger RNA(mRNA) encoding such proteins and ultimately to modulate the amount ofsuch proteins transcribed from their respective genes. Suchhybridization with mRNA interferes with the normal role of mRNA andcauses a modulation of its function in cells. The functions of mRNA tobe interfered with include all vital functions such as translocation ofthe RNA to the site for protein translation, actual translation ofprotein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and possibly even independent catalytic activity which may beengaged in by the RNA. The overall effect of such interference with mRNAfunction is modulation of the expression of a B7 protein, wherein“modulation” means either an increase (stimulation) or a decrease(inhibition) in the expression of a B7 protein. In the context of thepresent invention, inhibition is the preferred form of modulation ofgene expression.

Oligonucleotides may comprise nucleotide sequences sufficient inidentity and number to effect specific hybridization with a particularnucleic acid. Such oligonucleotides which specifically hybridize to aportion of the sense strand of a gene are commonly described as“antisense.” Antisense oligonucleotides are commonly used as researchreagents, diagnostic aids, and therapeutic agents. For example,antisense oligonucleotides, which are able to inhibit gene expressionwith exquisite specificity, are often used by those of ordinary skill toelucidate the function of particular genes, for example to distinguishbetween the functions of various members of a biological pathway. Thisspecific inhibitory effect has, therefore, been harnessed by thoseskilled in the art for research uses.

“Hybridization”, in the context of this invention, means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases, usually on oppositenucleic acid strands or two regions of a nucleic acid strand. Guanineand cytosine are examples of complementary bases which are known to formthree hydrogen bonds between them. Adenine and thymine are examples ofcomplementary bases which form two hydrogen bonds between them.“Specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of complementarity such that stable andspecific binding occurs between the DNA or RNA target and theoligonucleotide. It is understood that an oligonucleotide need not be100% complementary to its target nucleic acid sequence to bespecifically hybridizable. An oligonucleotide is specificallyhybridizable when binding of the oligonucleotide to the targetinterferes with the normal function of the target molecule to cause aloss of activity, and there is a sufficient degree of complementarity toavoid non-specific binding of the oligonucleotide to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment or, in the case of in vitro assays, underconditions in which the assays are conducted.

It is understood in the art that the sequence of the oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligomeric compound mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure or hairpin structure). It is preferred that the oligomericcompounds of the present invention comprise at least 70% sequencecomplementarity to a target region within the target nucleic acid, morepreferably that they comprise 90% sequence complementarity and even morepreferably comprise 95% sequence complementarity to the target regionwithin the target nucleic acid sequence to which they are targeted. Forexample, an oligomeric compound in which 18 of 20 nucleobases of theoligomeric compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an oligomeric compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an oligomeric compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656).

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which an oligomericcompound of the invention will hybridize to its target sequence, but toa minimal number of other sequences. Stringent conditions aresequence-dependent and will vary with different circumstances and in thecontext of this invention; “stringent conditions” under which oligomericcompounds hybridize to a target sequence are determined by the natureand composition of the oligomeric compounds and the assays in which theyare being investigated.

The specificity and sensitivity of oligonucleotides is also harnessed bythose of skill in the art for therapeutic uses. For example, thefollowing U.S. patents demonstrate palliative, therapeutic and othermethods utilizing antisense oligonucleotides. U.S. Pat. No. 5,135,917provides antisense oligonucleotides that inhibit human interleukin-1receptor expression. U.S. Pat. No. 5,098,890 is directed to antisenseoligonucleotides complementary to the c-myb oncogene and antisenseoligonucleotide therapies for certain cancerous conditions. U.S. Pat.No. 5,087,617 provides methods for treating cancer patients withantisense oligonucleotides. U.S. Pat. No. 5,166,195 providesoligonucleotide inhibitors of HIV. U.S. Pat. No. 5,004,810 providesoligomers capable of hybridizing to herpes simplex virus Vmw65 mRNA andinhibiting replication. U.S. Pat. No. 5,194,428 provides antisenseoligonucleotides having antiviral activity against influenza virus. U.S.Pat. No. 4,806,463 provides antisense oligonucleotides and methods usingthem to inhibit HTLV-III replication. U.S. Pat. No. 5,286,717 providesoligonucleotides having a complementary base sequence to a portion of anoncogene. U.S. Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423 aredirected to phosphorothioate oligonucleotide analogs used to preventreplication of foreign nucleic acids in cells. U.S. Pat. No. 4,689,320is directed to antisense oligonucleotides as antiviral agents specificto CMV. U.S. Pat. No. 5,098,890 provides oligonucleotides complementaryto at least a portion of the mRNA transcript of the human c-myb gene.U.S. Pat. No. 5,242,906 provides antisense oligonucleotides useful inthe treatment of latent EBV infections.

Oligonucleotides capable of modulating the expression of B7 proteinsrepresent a novel therapeutic class of anti-inflammatory agents withactivity towards a variety of inflammatory or autoimmune diseases, ordisorders or diseases with an inflammatory component such as asthma,juvenile diabetes mellitus, myasthenia gravis, Graves' disease,rheumatoid arthritis, allograft rejection, inflammatory bowel disease,multiple sclerosis, psoriasis, lupus erythematosus, systemic lupuserythematosus, diabetes, multiple sclerosis, contact dermatitis, eczema,atopic dermatitis, seborrheic dermatitis, nummular dermatitis,generalized exfoliative dermatitis, rhinitis and various allergies. Inaddition, oligonucleotides capable of modulating the expression of B7proteins provide a novel means of manipulating the ex vivo proliferationof T cells.

It is preferred to target specific genes for antisense attack.“Targeting” an oligonucleotide to the associated nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a foreign nucleic acid from aninfectious agent. In the present invention, the target is a cellulargene associated with several immune system disorders and diseases (suchas inflammation and autoimmune diseases), as well as with ostensibly“normal” immune reactions (such as a host animal's rejection oftransplanted tissue), for which modulation is desired in certaininstances. The targeting process also includes determination of a region(or regions) within this gene for the oligonucleotide interaction tooccur such that the desired effect, either detection or modulation ofexpression of the protein, will result. Once the target regions havebeen identified, oligonucleotides are chosen which are sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity to give the desired effect.

Generally, there are five regions of a gene that may be targeted forantisense modulation: the 5′ untranslated region (hereinafter, the“5′-UTR”), the translation initiation codon region (hereinafter, the“tIR”), the open reading frame (hereinafter, the “ORF”), the translationtermination codon region (hereinafter, the “tTR”) and the 3′untranslated region (hereinafter, the “3′-UTR”). As is known in the art,these regions are arranged in a typical messenger RNA molecule in thefollowing order (left to right, 5′ to 3′): 5′-UTR, tIR, ORF, tTR,3′-UTR. As is known in the art, although some eukaryotic transcripts aredirectly translated, many ORFs contain one or more sequences, known as“introns” which are excised from a transcript before it is translated;the expressed (unexcised) portions of the ORF are referred to as “exons”(Alberts et al., Molecular Biology of the Cell, 1983, Garland PublishingInc., New York, pp. 411-415). Furthermore, because many eukaryotic ORFsare a thousand nucleotides or more in length, it is often convenient tosubdivide the ORF into, e.g., the 5′ ORF region, the central ORF region,and the 3′ ORF region. In some instances, an ORF contains one or moresites that may be targeted due to some functional significance in vivo.Examples of the latter types of sites include intragenic stem-loopstructures (see, e.g., U.S. Pat. No. 5,512,438) and, in unprocessed mRNAmolecules, intron/exon splice sites. Within the context of the presentinvention, one preferred intragenic site is the region encompassing thetranslation initiation codon of the open reading frame (ORF) of thegene. Because, as is known in the art, the translation initiation codonis typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon.” A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Furthermore, 5′-UUU functions as atranslation initiation codon in vitro (Brigstock et al., Growth Factors,1990, 4, 45; Gelbert et al., Somat. Cell. Mol. Genet., 1990, 16, 173;Gold and Stormo, in: Escherichia coli and Salmonella typhimurium:Cellular and Molecular Biology, Vol. 2, 1987, Neidhardt et al., eds.,American Society for Microbiology, Washington, D.C., p. 1303). Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine(prokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions,in order to generate related polypeptides having different aminoterminal sequences (Markussen et al., Development, 1995, 121, 3723; Gaoet al., Cancer Res., 1995, 55, 743; McDermott et al, Gene, 1992, 117,193; Perri et al., J. Biol. Chem., 1991, 266, 12536; French et al., J.Virol., 1989, 63, 3270; Pushpa-Rekha et al., J. Biol. Chem., 1995, 270,26993; Monaco et al., J. Biol. Chem., 1994, 269, 347; DeVirgilio et al.,Yeast, 1992, 8, 1043; Kanagasundaram et al., Biochim. Biophys. Acta,1992, 1171, 198; Olsen et al., Mol. Endocrinol., 1991, 5, 1246; Saul etal., Appl. Environ. Microbiol., 1990, 56, 3117; Yaoita et al., Proc.Natl. Acad. Sci. USA, 1990, 87, 7090; Rogers et al., EMBO J., 1990, 9,2273). In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding a B7 protein, regardless of the sequence(s) of such codons. Itis also known in the art that a translation termination codon (or “stopcodon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAGand 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and5′-TGA, respectively). The terms “start codon region” and “translationinitiation region” refer to a portion of such an mRNA or gene thatencompasses from about 25 to about 50 contiguous nucleotides in eitherdirection (i.e., 5′ or 3′) from a translation initiation codon.Similarly, the terms “stop codon region” and “translation terminationregion” refer to a portion of such an mRNA or gene that encompasses fromabout 25 to about 50 contiguous nucleotides in either direction (i.e.,5′ or 3′) from a translation termination codon.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid or deoxyribonucleic acid.This term includes oligonucleotides composed of naturally-occurringnucleobases, sugars and covalent intersugar (backbone) linkages as wellas oligonucleotides having non-naturally-occurring portions whichfunction similarly. Such modified or substituted oligonucleotides areoften preferred over native forms because of desirable properties suchas, for example, enhanced cellular uptake, enhanced binding to targetand increased stability in the presence of nucleases.

While the preferred form of antisense compound is a single-strandedantisense oligonucleotide, in many species the introduction ofdouble-stranded structures, such as double-stranded RNA (dsRNA)molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

The first evidence that dsRNA could lead to gene silencing in animalscame in 1995 from work in the nematode, Caenorhabditis elegans (Guo andKempheus, Cell, 1995, 81, 611-620).

Montgomery et al. have shown that the primary interference effects ofdsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci.USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanismdefined in Caenorhabditis elegans resulting from exposure todouble-stranded RNA (dsRNA) has since been designated RNA interference(RNAi). This term has been generalized to mean antisense-mediated genesilencing involving the introduction of dsRNA leading to thesequence-specific reduction of endogenous targeted mRNA levels (Fire etal., Nature, 1998, 391, 806-811). Recently, it has been shown that itis, in fact, the single-stranded RNA oligomers of antisense polarity ofthe dsRNAs which are the potent inducers of RNAi (Tijsterman et al.,Science, 2002, 295, 694-697).

Oligomer and Monomer Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside linkage or in conjunctionwith the sugar ring the backbone of the oligonucleotide. The normalinternucleoside linkage that makes up the backbone of RNA and DNA is a3′ to 5′ phosphodiester linkage.

Modified Internucleoside Linkages

Specific examples of preferred antisense oligomeric compounds useful inthis invention include oligonucleotides containing modified e.g.non-naturally occurring internucleoside linkages. As defined in thisspecification, oligonucleotides having modified internucleoside linkagesinclude internucleoside linkages that retain a phosphorus atom andinternucleoside linkages that do not have a phosphorus atom. For thepurposes of this specification, and as sometimes referenced in the art,modified oligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone can also be considered to be oligonucleosides.

In the C. elegans system, modification of the internucleotide linkage(phosphorothioate) did not significantly interfere with RNAi activity.Based on this observation, it is suggested that certain preferredoligomeric compounds of the invention can also have one or more modifiedinternucleoside linkages. A preferred phosphorus containing modifiedinternucleoside linkage is the phosphorothioate internucleoside linkage.

Preferred modified oligonucleotide backbones containing a phosphorusatom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

In more preferred embodiments of the invention, oligomeric compoundshave one or more phosphorothioate and/or heteroatom internucleosidelinkages, in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—]. The MMI type internucleoside linkages aredisclosed in the above referenced U.S. Pat. No. 5,489,677. Preferredamide internucleoside linkages are disclosed in the above referencedU.S. Pat. No. 5,602,240.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Oligomer Mimetics

Another preferred group of oligomeric compounds amenable to the presentinvention includes oligonucleotide mimetics. The term mimetic as it isapplied to oligonucleotides is intended to include oligomeric compoundswherein only the furanose ring or both the furanose ring and theinternucleotide linkage are replaced with novel groups, replacement ofonly the furanose ring is also referred to in the art as being a sugarsurrogate. The heterocyclic base moiety or a modified heterocyclic basemoiety is maintained for hybridization with an appropriate targetnucleic acid. One such oligomeric compound, an oligonucleotide mimeticthat has been shown to have excellent hybridization properties, isreferred to as a peptide nucleic acid (PNA). In PNA oligomericcompounds, the sugar-backbone of an oligonucleotide is replaced with anamide containing backbone, in particular an aminoethylglycine backbone.The nucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. RepresentativeUnited States patents that teach the preparation of PNA oligomericcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA oligomeric compounds can be found inNielsen et al., Science, 1991, 254, 1497-1500.

One oligonucleotide mimetic that has been reported to have excellenthybridization properties is peptide nucleic acids (PNA). The backbone inPNA compounds is two or more linked aminoethylglycine units which givesPNA an amide containing backbone. The heterocyclic base moieties arebound directly or indirectly to aza nitrogen atoms of the amide portionof the backbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

PNA has been modified to incorporate numerous modifications since thebasic PNA structure was first prepared. The basic structure is shownbelow:

wherein

Bx is a heterocyclic base moiety;

T₄ is hydrogen, an amino protecting group, —C(O)R₅, substituted orunsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl,substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl,arylsulfonyl, a chemical functional group, a reporter group, a conjugategroup, a D or L α-amino acid linked via the α-carboxyl group oroptionally through the ω-carboxyl group when the amino acid is asparticacid or glutamic acid or a peptide derived from D, L or mixed D and Lamino acids linked through a carboxyl group, wherein the substituentgroups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl;

T₅ is —OH, —N(Z₁)Z₂, R₅, D or L α-amino acid linked via the α-aminogroup or optionally through the ω-amino group when the amino acid islysine or ornithine or a peptide derived from D, L or mixed D and Lamino acids linked through an amino group, a chemical functional group,a reporter group or a conjugate group;

Z₁ is hydrogen, C₁-C₆ alkyl, or an amino protecting group;

Z₂ is hydrogen, C₁-C₆ alkyl, an amino protecting group,—C(═O)—(CH₂)_(n)-J-Z₃, a D or L α-amino acid linked via the α-carboxylgroup or optionally through the ω-carboxyl group when the amino acid isaspartic acid or glutamic acid or a peptide derived from D, L or mixed Dand L amino acids linked through a carboxyl group;

Z₃ is hydrogen, an amino protecting group, —C₁-C₆ alkyl, —C(═O)—CH₃,benzyl, benzoyl, or —(CH₂)_(n)—N(H)Z₁;

each J is O, S or NH;

R₅ is a carbonyl protecting group; and

n is from 2 to about 50.

Another class of oligonucleotide mimetic that has been studied is basedon linked morpholino units (morpholino nucleic acid) having heterocyclicbases attached to the morpholino ring. A number of linking groups havebeen reported that link the morpholino monomeric units in a morpholinonucleic acid. A preferred class of linking groups have been selected togive a non-ionic oligomeric compound. The non-ionic morpholino-basedoligomeric compounds are less likely to have undesired interactions withcellular proteins. Morpholino-based oligomeric compounds are non-ionicmimics of oligonucleotides which are less likely to form undesiredinteractions with cellular proteins (Dwaine A. Braasch and David R.Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-basedoligomeric compounds are disclosed in U.S. Pat. No. 5,034,506, issuedJul. 23, 1991. The morpholino class of oligomeric compounds have beenprepared having a variety of different linking groups joining themonomeric subunits.

Morpholino nucleic acids have been prepared having a variety ofdifferent linking groups (L₂) joining the monomeric subunits. The basicformula is shown below:

wherein

T₁ is hydroxyl or a protected hydroxyl;

T₅ is hydrogen or a phosphate or phosphate derivative;

L₂ is a linking group; and

n is from 2 to about 50.

A further class of oligonucleotide mimetic is referred to ascyclohexenyl nucleic acids (CeNA). The furanose ring normally present ina DNA/RNA molecule is replaced with a cyclohexenyl ring. CeNA DMTprotected phosphoramidite monomers have been prepared and used foroligomeric compound synthesis following classical phosphoramiditechemistry. Fully modified CeNA oligomeric compounds and oligonucleotideshaving specific positions modified with CeNA have been prepared andstudied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). Ingeneral the incorporation of CeNA monomers into a DNA chain increasesits stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexeswith RNA and DNA complements with similar stability to the nativecomplexes. The study of incorporating CeNA structures into naturalnucleic acid structures was shown by NMR and circular dichroism toproceed with easy conformational adaptation. Furthermore theincorporation of CeNA into a sequence targeting RNA was stable to serumand able to activate E. Coli RNase resulting in cleavage of the targetRNA strand.

The general formula of CeNA is shown below:

wherein

each Bx is a heterocyclic base moiety;

T₁ is hydroxyl or a protected hydroxyl; and

T2 is hydroxyl or a protected hydroxyl.

Another class of oligonucleotide mimetic (anhydrohexitol nucleic acid)can be prepared from one or more anhydrohexitol nucleosides (see,Wouters and Herdewijn, Bioorg. Med. Chem. Lett., 1999, 9, 1563-1566) andwould have the general formula:

A further preferred modification includes Locked Nucleic Acids (LNAs) inwhich the 2′-hydroxyl group is linked to the 4′ carbon atom of the sugarring thereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming abicyclic sugar moiety. The linkage is preferably a methylene (—CH₂—)_(n)group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456). LNA and LNAanalogs display very high duplex thermal stabilities with complementaryDNA and RNA (Tm=+3 to +10 C), stability towards 3′-exonucleolyticdegradation and good solubility properties. The basic structure of LNAshowing the bicyclic ring system is shown below:

The conformations of LNAs determined by 2D NMR spectroscopy have shownthat the locked orientation of the LNA nucleotides, both insingle-stranded LNA and in duplexes, constrains the phosphate backbonein such a way as to introduce a higher population of the N-typeconformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53).These conformations are associated with improved stacking of thenucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18,1365-1370).

LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkinet al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNAhybridization was shown to be the most thermally stable nucleic acidtype duplex system, and the RNA-mimicking character of LNA wasestablished at the duplex level. Introduction of 3 LNA monomers (T or A)significantly increased melting points (Tm=+15/+11) toward DNAcomplements. The universality of LNA-mediated hybridization has beenstressed by the formation of exceedingly stable LNA:LNA duplexes. TheRNA-mimicking of LNA was reflected with regard to the N-typeconformational restriction of the monomers and to the secondarystructure of the LNA:RNA duplex.

LNAs also form duplexes with complementary DNA, RNA or LNA with highthermal affinities. Circular dichroism (CD) spectra show that duplexesinvolving fully modified LNA (esp. LNA:RNA) structurally resemble anA-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination ofan LNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer.Recognition of double-stranded DNA has also been demonstrated suggestingstrand invasion by LNA. Studies of mismatched sequences show that LNAsobey the Watson-Crick base pairing rules with generally improvedselectivity compared to the corresponding unmodified reference strands.

Novel types of LNA-oligomeric compounds, as well as the LNAs, are usefulin a wide range of diagnostic and therapeutic applications. Among theseare antisense applications, PCR applications, strand-displacementoligomers, substrates for nucleic acid polymerases and generally asnucleotide based drugs. Potent and nontoxic antisense oligonucleotidescontaining LNAs have been described (Wahlestedt et al., Proc. Natl.Acad. Sci. U.S.A., 2000, 97, 5633-5638.) The authors have demonstratedthat LNAs confer several desired properties to antisense agents. LNA/DNAcopolymers were not degraded readily in blood serum and cell extracts.LNA/DNA copolymers exhibited potent antisense activity in assay systemsas disparate as G-protein-coupled receptor signaling in living rat brainand detection of reporter genes in Escherichia coli. Lipofectin-mediatedefficient delivery of LNA into living human breast cancer cells has alsobeen accomplished.

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methyl-cytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

The first analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, havealso been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,2219-2222). Preparation of locked nucleoside analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., PCT InternationalApplication WO 98-DK393 19980914). Furthermore, synthesis of2′-amino-LNA, a novel conformationally restricted high-affinityoligonucleotide analog with a handle has been described in the art(Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition,2′-Amino- and 2′-methylamino-LNA's have been prepared and the thermalstability of their duplexes with complementary RNA and DNA strands hasbeen previously reported.

Further oligonucleotide mimetics have been prepared to include bicyclicand tricyclic nucleoside analogs having the formulas (amidite monomersshown):

(see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens etal., J. Am. Chem. Soc., 1999, 121, 3249-3255; and Renneberg et al., J.Am. Chem. Soc., 2002, 124, 5993-6002). These modified nucleoside analogshave been oligomerized using the phosphoramidite approach and theresulting oligomeric compounds containing tricyclic nucleoside analogshave shown increased thermal stabilities (Tm's) when hybridized to DNA,RNA and itself. Oligomeric compounds containing bicyclic nucleosideanalogs have shown thermal stabilities approaching that of DNA duplexes.

Another class of oligonucleotide mimetic referred to asphosphonomonoester nucleic acids incorporate a phosphorus group in thebackbone. This class of oligonucleotide mimetic is reported to haveuseful physical and biological and pharmacological properties in theareas of inhibiting gene expression (antisense oligonucleotides,ribozymes, sense oligonucleotides and triplex-forming oligonucleotides),as probes for the detection of nucleic acids and as auxiliaries for usein molecular biology.

The general formula (for definitions of Markush variables see: U.S. Pat.Nos. 5,874,553 and 6,127,346 herein incorporated by reference in theirentirety) is shown below.

Another oligonucleotide mimetic has been reported wherein the furanosylring has been replaced by a cyclobutyl moiety.

Modified Sugars

Oligomeric compounds of the invention may also contain one or moresubstituted sugar moieties. Preferred oligomeric compounds comprise asugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-,S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise a sugarsubstituent group selected from: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, amino alkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂.

Other preferred sugar substituent groups include methoxy (—O—CH₃),aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl(—O—CH₂—CH═CH₂) and fluoro (F). 2′-Sugar substituent groups may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligomeric compound, particularly the 3′position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligomeric compounds may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

Further representative sugar substituent groups include groups offormula I_(a) or II_(a):

wherein:

R_(b) is O, S or NH;

R_(d) is a single bond, O, S or C(═O);

R_(e) is C₁-C₁₀ alkyl, N(R_(k))(R_(m)), N(R_(k))(R_(n)),N═C(R_(p))(R_(q)), N═C(R_(p))(R_(r)) or has formula III_(a);

R_(p) and R_(q) are each independently hydrogen or C₁-C₁₀ alkyl;

R_(r) is —R_(x)—R_(y);

each R_(s), R_(t), R_(u) and R_(v) is, independently, hydrogen,C(O)R_(w), substituted or unsubstituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or aconjugate group, wherein the substituent groups are selected fromhydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

or optionally, R_(u) and R_(v), together form a phthalimido moiety withthe nitrogen atom to which they are attached;

each R_(w) is, independently, substituted or unsubstituted C₁-C₁₀ alkyl,trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy,9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy,benzyloxy, butyryl, iso-butyryl, phenyl or aryl;

R_(k) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);

R_(p) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);

R_(x) is a bond or a linking moiety;

R_(y) is a chemical functional group, a conjugate group or a solidsupport medium;

each R_(m) and R_(n) is, independently, H, a nitrogen protecting group,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, wherein thesubstituent groups are selected from hydroxyl, amino, alkoxy, carboxy,benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl,alkynyl; NH₃ ⁺, N(R_(u))(R_(v)), guanidino and acyl where said acyl isan acid amide or an ester;

or R_(m) and R_(n), together, are a nitrogen protecting group, arejoined in a ring structure that optionally includes an additionalheteroatom selected from N and O or are a chemical functional group;

R_(i) is OR_(z), SR_(z), or N(R_(z))₂;

each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(H)R_(u), C(═O)N(H)R_(u) or OC(═O)N(H)R_(u);

R_(f), R_(g) and R_(h) comprise a ring system having from about 4 toabout 7 carbon atoms or having from about 3 to about 6 carbon atoms and1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen,nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic;

R_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenylhaving 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbonatoms, aryl having 6 to about 14 carbon atoms, N(R_(k))(R_(m))OR_(k),halo, SR_(k) or CN;

m_(a) is 1 to about 10;

each mb is, independently, 0 or 1;

mc is 0 or an integer from 1 to 10;

md is an integer from 1 to 10;

me is from 0, 1 or 2; and

provided that when mc is 0, md is greater than 1.

Representative substituents groups of Formula I are disclosed in U.S.patent application Ser. No. 09/130,973, filed Aug. 7, 1998, entitled“Capped 2′-Oxyethoxy Oligonucleotides,” hereby incorporated by referencein its entirety.

Representative cyclic substituent groups of Formula II are disclosed inU.S. patent application Ser. No. 09/123,108, filed Jul. 27, 1998,entitled “RNA Targeted 2′-Oligomeric compounds that are ConformationallyPreorganized,” hereby incorporated by reference in its entirety.

Particularly preferred sugar substituent groups includeO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10.

Representative guanidino substituent groups that are shown in formulaIII and IV are disclosed in co-owned U.S. patent application Ser. No.09/349,040, entitled “Functionalized Oligomers”, filed Jul. 7, 1999,hereby incorporated by reference in its entirety.

Representative acetamido substituent groups are disclosed in U.S. Pat.No. 6,147,200 which is hereby incorporated by reference in its entirety.

Representative dimethylaminoethyloxyethyl substituent groups aredisclosed in International Patent Application PCT/US99/17895, entitled“2′-O-Dimethylaminoethyloxyethyl-Oligomeric compounds”, filed Aug. 6,1999, hereby incorporated by reference in its entirety.

Modified Nucleobases/Naturally Occurring Nucleobases

Oligomeric compounds may also include nucleobase (often referred to inthe art simply as “base” or “heterocyclic base moiety”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesalso referred herein as heterocyclic base moieties include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

In one aspect of the present invention oligomeric compounds are preparedhaving polycyclic heterocyclic compounds in place of one or moreheterocyclic base moieties. A number of tricyclic heterocyclic compoundshave been previously reported. These compounds are routinely used inantisense applications to increase the binding properties of themodified strand to a target strand. The most studied modifications aretargeted to guanosines hence they have been termed G-clamps or cytidineanalogs. Many of these polycyclic heterocyclic compounds have thegeneral formula:

Representative cytosine analogs that make 3 hydrogen bonds with aguanosine in a second strand include 1,3-diazaphenoxazine-2-one (R₁₀═O,R₁₁-R₁₄═H) [Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16,1837-1846], 1,3-diazaphenothiazine-2-one (R₁₀₌S, R₁₁-R₁₄═H), [Lin,K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117,3873-3874] and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀═O,R₁₁-R₁₄═F) [Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998,39, 8385-8388]. Incorporated into oligonucleotides these basemodifications were shown to hybridize with complementary guanine and thelatter was also shown to hybridize with adenine and to enhance helicalthermal stability by extended stacking interactions (also see U.S.patent application entitled “Modified Peptide Nucleic Acids” filed May24, 2002, Ser. No. 10/155,920; and U.S. patent application entitled“Nuclease Resistant Chimeric Oligonucleotides” filed May 24, 2002, Ser.No. 10/013,295, both of which are commonly owned with this applicationand are herein incorporated by reference in their entirety).

Further helix-stabilizing properties have been observed when a cytosineanalog/substitute has an aminoethoxy moiety attached to the rigid1,3-diazaphenoxazine-2-one scaffold (R₁₀═O, R₁₁═—O—(CH₂)₂—NH₂, R₁₂₋₁₄═H)[Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532].Binding studies demonstrated that a single incorporation could enhancethe binding affinity of a model oligonucleotide to its complementarytarget DNA or RNA with a ΔT_(m) of up to 18° relative to 5-methylcytosine (dC5^(me)), which is the highest known affinity enhancement fora single modification, yet. On the other hand, the gain in helicalstability does not compromise the specificity of the oligonucleotides.The T_(m) data indicate an even greater discrimination between theperfect match and mismatched sequences compared to dC5^(me). It wassuggested that the tethered amino group serves as an additional hydrogenbond donor to interact with the Hoogsteen face, namely the O6, of acomplementary guanine thereby forming 4 hydrogen bonds. This means thatthe increased affinity of G-clamp is mediated by the combination ofextended base stacking and additional specific hydrogen bonding.

Further tricyclic heterocyclic compounds and methods of using them thatare amenable to the present invention are disclosed in U.S. Pat. No.6,028,183, which issued on May 22, 2000, and U.S. Pat. No. 6,007,992,which issued on Dec. 28, 1999, the contents of both are commonlyassigned with this application and are incorporated herein in theirentirety.

The enhanced binding affinity of the phenoxazine derivatives togetherwith their uncompromised sequence specificity makes them valuablenucleobase analogs for the development of more potent antisense-baseddrugs. In fact, promising data have been derived from in vitroexperiments demonstrating that heptanucleotides containing phenoxazinesubstitutions are capable to activate RNaseH, enhance cellular uptakeand exhibit an increased antisense activity [Lin, K-Y; Matteucci, M. J.Am. Chem. Soc. 1998, 120, 8531-8532]. The activity enhancement was evenmore pronounced in case of G-clamp, as a single substitution was shownto significantly improve the in vitro potency of a 20mer2′-deoxyphosphorothioate oligonucleotides [Flanagan, W. M.; Wolf, J. J.;Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc.Natl. Acad. Sci. USA, 1999, 96, 3513-3518]. Nevertheless, to optimizeoligonucleotide design and to better understand the impact of theseheterocyclic modifications on the biological activity, it is importantto evaluate their effect on the nuclease stability of the oligomers.

Further modified polycyclic heterocyclic compounds useful asheterocyclic bases are disclosed in but not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692;5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. patentapplication Ser. No. 09/996,292 filed Nov. 28, 2001, certain of whichare commonly owned with the instant application, and each of which isherein incorporated by reference.

The oligonucleotides of the present invention also include variants inwhich a different base is present at one or more of the nucleotidepositions in the oligonucleotide. For example, if the first nucleotideis an adenosine, variants may be produced which contain thymidine,guanosine or cytidine at this position. This may be done at any of thepositions of the oligonucleotide. Thus, a 20-mer may comprise 60variations (20 positions×3 alternates at each position) in which theoriginal nucleotide is substituted with any of the three alternatenucleotides. These oligonucleotides are then tested using the methodsdescribed herein to determine their ability to inhibit expression of HCVmRNA and/or HCV replication.

Conjugates

A further preferred substitution that can be appended to the oligomericcompounds of the invention involves the linkage of one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the resulting oligomeric compounds. In one embodimentsuch modified oligomeric compounds are prepared by covalently attachingconjugate groups to functional groups such as hydroxyl or amino groups.Conjugate groups of the invention include intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, polyethers,groups that enhance the pharmacodynamic properties of oligomers, andgroups that enhance the pharmacokinetic properties of oligomers. Typicalconjugates groups include cholesterols, lipids, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties, in the context of this invention, includegroups that improve oligomer uptake, enhance oligomer resistance todegradation, and/or strengthen sequence-specific hybridization with RNA.Groups that enhance the pharmacokinetic properties, in the context ofthis invention, include groups that improve oligomer uptake,distribution, metabolism or excretion. Representative conjugate groupsare disclosed in International Patent Application PCT/US92/09196, filedOct. 23, 1992 the entire disclosure of which is incorporated herein byreference. Conjugate moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al.,Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

The oligomeric compounds of the invention may also be conjugated toactive drug substances, for example, aspirin, warfarin, phenylbutazone,ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen,carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indomethicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

Chimeric Oligomeric Compounds

It is not necessary for all positions in an oligomeric compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligomeric compound oreven at a single monomeric subunit such as a nucleoside within anoligomeric compound. The present invention also includes oligomericcompounds which are chimeric oligomeric compounds. “Chimeric” oligomericcompounds or “chimeras,” in the context of this invention, areoligomeric compounds that contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a nucleic acid based oligomer.

Chimeric oligomeric compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligomeric compound mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of inhibition of gene expression. Consequently,comparable results can often be obtained with shorter oligomericcompounds when chimeras are used, compared to for examplephosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

Chimeric oligomeric compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, oligonucleotideanalogs, oligonucleosides and/or oligonucleotide mimetics as describedabove. Such oligomeric compounds have also been referred to in the artas hybrids hemimers, gapmers or inverted gapmers. Representative UnitedStates patents that teach the preparation of such hybrid structuresinclude, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797;5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350;5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

3′-Endo Modifications

In one aspect of the present invention oligomeric compounds includenucleosides synthetically modified to induce a 3′-endo sugarconformation. A nucleoside can incorporate synthetic modifications ofthe heterocyclic base, the sugar moiety or both to induce a desired3′-endo sugar conformation. These modified nucleosides are used to mimicRNA like nucleosides so that particular properties of an oligomericcompound can be enhanced while maintaining the desirable 3′-endoconformational geometry. There is an apparent preference for an RNA typeduplex (A form helix, predominantly 3′-endo) as a requirement (e.g.trigger) of RNA interference which is supported in part by the fact thatduplexes composed of 2′-deoxy-2′-F-nucleosides appear efficient intriggering RNAi response in the C. elegans system. Properties that areenhanced by using more stable 3′-endo nucleosides include but aren'tlimited to modulation of pharmacokinetic properties through modificationof protein binding, protein off-rate, absorption and clearance;modulation of nuclease stability as well as chemical stability;modulation of the binding affinity and specificity of the oligomer(affinity and specificity for enzymes as well as for complementarysequences); and increasing efficacy of RNA cleavage. The presentinvention provides oligomeric triggers of RNAi having one or morenucleosides modified in such a way as to favor a C3′-endo typeconformation.

Nucleoside conformation is influenced by various factors includingsubstitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar.Electronegative substituents generally prefer the axial positions, whilesterically demanding substituents generally prefer the equatorialpositions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984,Springer-Verlag.) Modification of the 2′ position to favor the 3′-endoconformation can be achieved while maintaining the 2′-OH as arecognition element, as illustrated in FIG. 2, below (Gallo et al.,Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem.,(1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64,747-754.) Alternatively, preference for the 3′-endo conformation can beachieved by deletion of the 2′-OH as exemplified by2′deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36,831-841), which adopts the 3′-endo conformation positioning theelectronegative fluorine atom in the axial position. Other modificationsof the ribose ring, for example substitution at the 4′-position to give4′-F modified nucleosides (Guillerm et al., Bioorganic and MedicinalChemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem.(1976), 41, 3010-3017), or for example modification to yieldmethanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett.(2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal ChemistryLetters (2001), 11, 1333-1337) also induce preference for the 3′-endoconformation. Along similar lines, oligomeric triggers of RNAi responsemight be composed of one or more nucleosides modified in such a way thatconformation is locked into a C3′-endo type conformation, i.e. LockedNucleic Acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), andethylene bridged Nucleic Acids (ENA, Morita et al, Bioorganic &Medicinal Chemistry Letters (2002), 12, 73-76.) Examples of modifiednucleosides amenable to the present invention are shown below in TableI. These examples are meant to be representative and not exhaustive.

TABLE I

The preferred conformation of modified nucleosides and their oligomerscan be estimated by various methods such as molecular dynamicscalculations, nuclear magnetic resonance spectroscopy and CDmeasurements. Hence, modifications predicted to induce RNA likeconformations, A-form duplex geometry in an oligomeric context, areselected for use in the modified oligonucleotides of the presentinvention. The synthesis of numerous of the modified nucleosidesamenable to the present invention are known in the art (see for example,Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend,1988, Plenum press., and the examples section below.) Nucleosides knownto be inhibitors/substrates for RNA dependent RNA polymerases (forexample HCV NS5B).

In one aspect, the present invention is directed to oligonucleotidesthat are prepared having enhanced properties compared to native RNAagainst nucleic acid targets. A target is identified and anoligonucleotide is selected having an effective length and sequence thatis complementary to a portion of the target sequence. Each nucleoside ofthe selected sequence is scrutinized for possible enhancingmodifications. A preferred modification would be the replacement of oneor more RNA nucleosides with nucleosides that have the same 3′-endoconformational geometry. Such modifications can enhance chemical andnuclease stability relative to native RNA while at the same time beingmuch cheaper and easier to synthesize and/or incorporate into anoligonucleotide. The selected sequence can be further divided intoregions and the nucleosides of each region evaluated for enhancingmodifications that can be the result of a chimeric configuration.Consideration is also given to the 5′ and 3′-termini as there are oftenadvantageous modifications that can be made to one or more of theterminal nucleosides. The oligomeric compounds of the present inventioninclude at least one 5′-modified phosphate group on a single strand oron at least one 5′-position of a double stranded sequence or sequences.Further modifications are also considered such as internucleosidelinkages, conjugate groups, substitute sugars or bases, substitution ofone or more nucleosides with nucleoside mimetics and any othermodification that can enhance the selected sequence for its intendedtarget. The terms used to describe the conformational geometry ofhomoduplex nucleic acids are “A Form” for RNA and “B Form” for DNA. Therespective conformational geometry for RNA and DNA duplexes wasdetermined from X-ray diffraction analysis of nucleic acid fibers(Arnott and Hukins, Biochem. Biophys. Res. Comm., 1970, 47, 1504.) Ingeneral, RNA:RNA duplexes are more stable and have higher meltingtemperatures (Tm's) than DNA:DNA duplexes (Sanger et al., Principles ofNucleic Acid Structure, 1984, Springer-Verlag; New York, N.Y.; Lesnik etal., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic AcidsRes., 1997, 25, 2627-2634). The increased stability of RNA has beenattributed to several structural features, most notably the improvedbase stacking interactions that result from an A-form geometry (Searleet al., Nucleic Acids Res., 1993, 21, 2051-2056). The presence of the 2′hydroxyl in RNA biases the sugar toward a C3′ endo pucker, i.e., alsodesignated as Northern pucker, which causes the duplex to favor theA-form geometry. In addition, the 2′ hydroxyl groups of RNA can form anetwork of water mediated hydrogen bonds that help stabilize the RNAduplex (Egli et al., Biochemistry, 1996, 35, 8489-8494). On the otherhand, deoxy nucleic acids prefer a C2′ endo sugar pucker, i.e., alsoknown as Southern pucker, which is thought to impart a less stableB-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure,Springer-Verlag, New York, N.Y.). As used herein, B-form geometry isinclusive of both C2′-endo pucker and O4′-endo pucker. This isconsistent with Berger, et. al., Nucleic Acids Research, 1998, 26,2473-2480, who pointed out that in considering the furanoseconformations which give rise to B-form duplexes consideration shouldalso be given to a O4′-endo pucker contribution.

DNA:RNA hybrid duplexes, however, are usually less stable than pureRNA:RNA duplexes, and depending on their sequence may be either more orless stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res.,1993, 21, 2051-2056). The structure of a hybrid duplex is intermediatebetween A- and B-form geometries, which may result in poor stackinginteractions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306;Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al.,Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996,264, 521-533). The stability of the duplex formed between a target RNAand a synthetic sequence is central to therapies such as but not limitedto antisense and RNA interference as these mechanisms require thebinding of a synthetic oligonucleotide strand to an RNA target strand.In the case of antisense, effective inhibition of the mRNA requires thatthe antisense DNA have a very high binding affinity with the mRNA.Otherwise the desired interaction between the synthetic oligonucleotidestrand and target mRNA strand will occur infrequently, resulting indecreased efficacy.

One routinely used method of modifying the sugar puckering is thesubstitution of the sugar at the 2′-position with a substituent groupthat influences the sugar geometry. The influence on ring conformationis dependant on the nature of the substituent at the 2′-position. Anumber of different substituents have been studied to determine theirsugar puckering effect. For example, 2′-halogens have been studiedshowing that the 2′-fluoro derivative exhibits the largest population(65%) of the C3′-endo form, and the 2′-iodo exhibits the lowestpopulation (7%). The populations of adenosine (2′-OH) versusdeoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, theeffect of the 2′-fluoro group of adenosine dimers(2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is furthercorrelated to the stabilization of the stacked conformation.

As expected, the relative duplex stability can be enhanced byreplacement of 2′-OH groups with 2′-F groups thereby increasing theC3′-endo population. It is assumed that the highly polar nature of the2′-F bond and the extreme preference for C3′-endo puckering maystabilize the stacked conformation in an A-form duplex. Data from UVhypochromicity, circular dichroism, and ¹H NMR also indicate that thedegree of stacking decreases as the electronegativity of the halosubstituent decreases. Furthermore, steric bulk at the 2′-position ofthe sugar moiety is better accommodated in an A-form duplex than aB-form duplex. Thus, a 2′-substituent on the 3′-terminus of adinucleoside monophosphate is thought to exert a number of effects onthe stacking conformation: steric repulsion, furanose puckeringpreference, electrostatic repulsion, hydrophobic attraction, andhydrogen bonding capabilities. These substituent effects are thought tobe determined by the molecular size, electronegativity, andhydrophobicity of the substituent. Melting temperatures of complementarystrands is also increased with the 2′-substituted adenosinediphosphates. It is not clear whether the 3′-endo preference of theconformation or the presence of the substituent is responsible for theincreased binding. However, greater overlap of adjacent bases (stacking)can be achieved with the 3′-endo conformation.

One synthetic 2′-modification that imparts increased nuclease resistanceand a very high binding affinity to nucleotides is the 2-methoxyethoxy(2′-MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997,272, 11944-12000). One of the immediate advantages of the 2′-MOEsubstitution is the improvement in binding affinity, which is greaterthan many similar 2′ modifications such as O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-O-methoxyethyl substituentalso have been shown to be antisense inhibitors of gene expression withpromising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995,78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al.,Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., NucleosidesNucleotides, 1997, 16, 917-926). Relative to DNA, the oligonucleotideshaving the 2′-MOE modification displayed improved RNA affinity andhigher nuclease resistance. Chimeric oligonucleotides having 2′-MOEsubstituents in the wing nucleosides and an internal region ofdeoxy-phosphorothioate nucleotides (also termed a gapped oligonucleotideor gapmer) have shown effective reduction in the growth of tumors inanimal models at low doses. 2′-MOE substituted oligonucleotides havealso shown outstanding promise as antisense agents in several diseasestates. One such MOE substituted oligonucleotide is presently beinginvestigated in clinical trials for the treatment of CMV retinitis.

Chemistries Defined

Unless otherwise defined herein, alkyl means C₁-C₁₂, preferably C₁-C₈,and more preferably C₁-C₆, straight or (where possible) branched chainaliphatic hydrocarbyl.

Unless otherwise defined herein, heteroalkyl means C₁-C₁₂, preferablyC₁-C₈, and more preferably C₁-C₆, straight or (where possible) branchedchain aliphatic hydrocarbyl containing at least one, and preferablyabout 1 to about 3, hetero atoms in the chain, including the terminalportion of the chain. Preferred heteroatoms include N, O and S.

Unless otherwise defined herein, cycloalkyl means C₃-C₁₂, preferablyC₃-C₈, and more preferably C₃-C₆, aliphatic hydrocarbyl ring.

Unless otherwise defined herein, alkenyl means C₂-C₁₂, preferably C₂-C₈,and more preferably C₂-C₆ alkenyl, which may be straight or (wherepossible) branched hydrocarbyl moiety, which contains at least onecarbon-carbon double bond.

Unless otherwise defined herein, alkynyl means C₂-C₁₂, preferably C₂-C₈,and more preferably C₂-C₆ alkynyl, which may be straight or (wherepossible) branched hydrocarbyl moiety, which contains at least onecarbon-carbon triple bond.

Unless otherwise defined herein, heterocycloalkyl means a ring moietycontaining at least three ring members, at least one of which is carbon,and of which 1, 2 or three ring members are other than carbon.Preferably the number of carbon atoms varies from 1 to about 12,preferably 1 to about 6, and the total number of ring members variesfrom three to about 15, preferably from about 3 to about 8. Preferredring heteroatoms are N, O and S. Preferred heterocycloalkyl groupsinclude morpholino, thiomorpholino, piperidinyl, piperazinyl,homopiperidinyl, homopiperazinyl, homomorpholino, homothiomorpholino,pyrrolodinyl, tetrahydrooxazolyl, tetrahydroimidazolyl,tetrahydrothiazolyl, tetrahydroisoxazolyl, tetrahydropyrrazolyl,furanyl, pyranyl, and tetrahydroisothiazolyl.

Unless otherwise defined herein, aryl means any hydrocarbon ringstructure containing at least one aryl ring. Preferred aryl rings haveabout 6 to about 20 ring carbons. Especially preferred aryl ringsinclude phenyl, napthyl, anthracenyl, and phenanthrenyl.

Unless otherwise defined herein, hetaryl means a ring moiety containingat least one fully unsaturated ring, the ring consisting of carbon andnon-carbon atoms. Preferably the ring system contains about 1 to about 4rings. Preferably the number of carbon atoms varies from 1 to about 12,preferably 1 to about 6, and the total number of ring members variesfrom three to about 15, preferably from about 3 to about 8. Preferredring heteroatoms are N, O and S. Preferred hetaryl moieties includepyrazolyl, thiophenyl, pyridyl, imidazolyl, tetrazolyl, pyridyl,pyrimidinyl, purinyl, quinazolinyl, quinoxalinyl, benzimidazolyl,benzothiophenyl, etc.

Unless otherwise defined herein, where a moiety is defined as a compoundmoiety, such as hetarylalkyl (hetaryl and alkyl), aralkyl (aryl andalkyl), etc., each of the sub-moieties is as defined herein.

Unless otherwise defined herein, an electron withdrawing group is agroup, such as the cyano or isocyanato group that draws electroniccharge away from the carbon to which it is attached. Other electronwithdrawing groups of note include those whose electronegativitiesexceed that of carbon, for example halogen, nitro, or phenyl substitutedin the ortho- or para-position with one or more cyano, isothiocyanato,nitro or halo groups.

Unless otherwise defined herein, the terms halogen and halo have theirordinary meanings. Preferred halo (halogen) substituents are Cl, Br, andI.

The aforementioned optional substituents are, unless otherwise hereindefined, suitable substituents depending upon desired properties.Included are halogens (Cl, Br, I), alkyl, alkenyl, and alkynyl moieties,NO₂, NH₃ (substituted and unsubstituted), acid moieties (e.g. —CO₂H,—OSO₃H₂, etc.), heterocycloalkyl moieties, hetaryl moieties, arylmoieties, etc.

In all the preceding formulae, the squiggle (˜) indicates a bond to anoxygen or sulfur of the 5′-phosphate.

Phosphate protecting groups include those described in U.S. Pat. No.5,760,209, U.S. Pat. No. 5,614,621, U.S. Pat. No. 6,051,699, U.S. Pat.No. 6,020,475, U.S. Pat. No. 6,326,478, U.S. Pat. No. 6,169,177, U.S.Pat. No. 6,121,437, U.S. Pat. No. 6,465,628 each of which is expresslyincorporated herein by reference in its entirety.

The oligonucleotides in accordance with this invention (single strandedor double stranded) preferably comprise from about 8 to about 80nucleotides, more preferably from about 12-50 nucleotides and mostpreferably from about 15 to 30 nucleotides. As is known in the art, anucleotide is a base-sugar combination suitably bound to an adjacentnucleotide through a phosphodiester, phosphorothioate or other covalentlinkage.

The oligonucleotides of the present invention also include variants inwhich a different base is present at one or more of the nucleotidepositions in the oligonucleotide. For example, if the first nucleotideis an adenosine, variants may be produced which contain thymidine,guanosine or cytidine at this position. This may be done at any of thepositions of the oligonucleotide. Thus, a 20-mer may comprise 60variations (20 positions×3 alternates at each position) in which theoriginal nucleotide is substituted with any of the three alternatenucleotides. These oligonucleotides are then tested using the methodsdescribed herein to determine their ability to inhibit expression ofB7.1 or B7.2 mRNA.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives.

The oligonucleotides of the present invention can be utilized astherapeutic compounds, diagnostic tools and as research reagents andkits. The term “therapeutic uses” is intended to encompass prophylactic,palliative and curative uses wherein the oligonucleotides of theinvention are contacted with animal cells either in vivo or ex vivo.When contacted with animal cells ex vivo, a therapeutic use includesincorporating such cells into an animal after treatment with one or moreoligonucleotides of the invention. While not intending to be bound to aparticular utility, the ex vivo modulation of, e.g., T cellproliferation by the oligonucleotides of the invention can be employedin, for example, potential therapeutic modalities wherein it is desiredto modulate the expression of a B7 protein in APCs.

As an example, oligonucleotides that inhibit the expression of B7-1proteins are expected to enhance the availability of B7-2 proteins onthe surface of APCs, thus increasing the costimulatory effect of B7-2 onT cells ex vivo (Levine et al., Science, 1996, 272, 1939).

For therapeutic uses, an animal suspected of having a disease ordisorder which can be treated or prevented by modulating the expressionor activity of a B7 protein is, for example, treated by administeringoligonucleotides in accordance with this invention. The oligonucleotidesof the invention can be utilized in pharmaceutical compositions byadding an effective amount of an oligonucleotide to a suitablepharmaceutically acceptable diluent or carrier. Workers in the fieldhave identified antisense, triplex and other oligonucleotidecompositions which are capable of modulating expression of genesimplicated in viral, fungal and metabolic diseases. Antisenseoligonucleotides have been safely administered to humans and severalclinical trials are presently underway. It is thus established thatoligonucleotides can be useful therapeutic instrumentalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans.

The oligonucleotides of the present invention can be further used todetect the presence of B7-specific nucleic acids in a cell or tissuesample. For example, radiolabeled oligonucleotides can be prepared by³²P labeling at the 5′ end with polynucleotide kinase (Sambrook et al.,Molecular Cloning. A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989, Volume 2, pg. 10.59). Radiolabeled oligonucleotides arethen contacted with cell or tissue samples suspected of containing B7message RNAs (and thus B7 proteins), and the samples are washed toremove unbound oligonucleotide. Radioactivity remaining in the sampleindicates the presence of bound oligonucleotide, which in turn indicatesthe presence of nucleic acids complementary to the oligonucleotide, andcan be quantitated using a scintillation counter or other routine means.Expression of nucleic acids encoding these proteins is thus detected.

Radiolabeled oligonucleotides of the present invention can also be usedto perform autoradiography of tissues to determine the localization,distribution and quantitation of B7 proteins for research, diagnostic ortherapeutic purposes. In such studies, tissue sections are treated withradiolabeled oligonucleotide and washed as described above, then exposedto photographic emulsion according to routine autoradiographyprocedures. The emulsion, when developed, yields an image of silvergrains over the regions expressing a B7 gene. Quantitation of the silvergrains permits detection of the expression of mRNA molecules encodingthese proteins and permits targeting of oligonucleotides to these areas.

Analogous assays for fluorescent detection of expression of B7 nucleicacids can be developed using oligonucleotides of the present inventionwhich are conjugated with fluorescein or other fluorescent tags insteadof radiolabeling. Such conjugations are routinely accomplished duringsolid phase synthesis using fluorescently-labeled amidites or controlledpore glass (CPG) columns. Fluorescein-labeled amidites and CPG areavailable from, e.g., Glen Research, Sterling Va.

The present invention employs oligonucleotides targeted to nucleic acidsencoding B7 proteins and oligonucleotides targeted to nucleic acidsencoding such proteins. Kits for detecting the presence or absence ofexpression of a B7 protein may also be prepared. Such kits include anoligonucleotide targeted to an appropriate gene, i.e., a gene encoding aB7 protein. Appropriate kit and assay formats, such as, e.g., “sandwich”assays, are known in the art and can easily be adapted for use with theoligonucleotides of the invention. Hybridization of the oligonucleotidesof the invention with a nucleic acid encoding a B7 protein can bedetected by means known in the art. Such means may include conjugationof an enzyme to the oligonucleotide, radiolabeling of theoligonucleotide or any other suitable detection systems. Kits fordetecting the presence or absence of a B7 protein may also be prepared.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleotides. For example,adenine and thymine are complementary nucleobases which pair through theformation of hydrogen bonds. “Complementary,” as used herein, refers tothe capacity for precise pairing between two nucleotides. For example,if a nucleotide at a certain position of an oligonucleotide is capableof hydrogen bonding with a nucleotide at the same position of a DNA orRNA molecule, then the oligonucleotide and the DNA or RNA are consideredto be complementary to each other at that position. The oligonucleotideand the DNA or RNA are complementary to each other when a sufficientnumber of corresponding positions in each molecule are occupied bynucleotides which can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of complementarity or precise pairing such that stableand specific binding occurs between the oligonucleotide and the DNA orRNA target. It is understood in the art that an oligonucleotide need notbe 100% complementary to its target DNA sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target DNA or RNA moleculeinterferes with the normal function of the target DNA or RNA to cause adecrease or loss of function, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, or in the case of in vitro assays,under conditions in which the assays are performed.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.In general, for therapeutics, a patient in need of such therapy isadministered an oligonucleotide in accordance with the invention,commonly in a pharmaceutically acceptable carrier, in doses ranging from0.01 μg to 100 g per kg of body weight depending on the age of thepatient and the severity of the disorder or disease state being treated.Further, the treatment regimen may last for a period of time which willvary depending upon the nature of the particular disease or disorder,its severity and the overall condition of the patient, and may extendfrom once daily to once every 20 years. Following treatment, the patientis monitored for changes in his/her condition and for alleviation of thesymptoms of the disorder or disease state. The dosage of theoligonucleotide may either be increased in the event the patient doesnot respond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disorder or diseasestate is observed, or if the disorder or disease state has been ablated.

In some cases, it may be more effective to treat a patient with anoligonucleotide of the invention in conjunction with other therapeuticmodalities in order to increase the efficacy of a treatment regimen. Inthe context of the invention, the term “a treatment regimen” is meant toencompass therapeutic, palliative and prophylactic modalities. In apreferred embodiment, the oligonucleotides of the invention are used inconjunction with an anti-inflammatory and/or immunosuppressive agent,preferably one or more antisense oligonucleotides targeted to anintercellular adhesion molecule (ICAM), preferably to ICAM-1. Otheranti-inflammatory and/or immunosuppressive agents that may be used incombination with the oligonucleotides of the invention include, but arenot limited to, soluble ICAM proteins (e.g., sICAM-1), antibody-toxinconjugates, prednisone, methylprednisolone, azathioprine,cyclophosphamide, cyclosporine, interferons, sympathomimetics,conventional antihistamines (histamine H₁ receptor antagonists,including, for example, brompheniramine maleate, chlorpheniraminemaleate, dexchlorpheniramine maleate, tripolidine HCl, carbinoxaminemaleate, clemastine fumarate, dimenhydrinate, diphenhydramine HCl,diphenylpyraline HCl, doxylamine succinate, tripelennamine citrate,tripelennamine HCl, cyclizine HCl, hydroxyzine HCl, meclizine HCl,methdilazine HCl, promethazine HCl, trimeprazine tartrate, azatadinemaleate, cyproheptadine HCl, terfenadine, etc.), histamine H₂ receptorantagonists (e.g., ranitidine). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 302-336 and 2516-2522). When used with the compounds of theinvention, such agents may be used individually, sequentially, or incombination with one or more other such agents.

In another preferred embodiment of the invention, an antisenseoligonucleotide targeted to one B7 mRNA species (e.g., B7-1) is used incombination with an antisense oligonucleotide targeted to a second B7mRNA species (e.g., B7-2) in order to inhibit the costimulatory effectof B7 molecules to a more extensive degree than can be achieved witheither oligonucleotide used individually. In a related version of thisembodiment, two or more oligonucleotides of the invention, each targetedto an alternatively spliced B7-1 or B7-2 mRNA, are combined with eachother in order to inhibit expression of both forms of the alternativelyspliced mRNAs. It is known in the art that, depending on the specificityof the modulating agent employed, inhibition of one form of analternatively spliced mRNA may not result in a sufficient reduction ofexpression for a given condition to be manifest. Thus, such combinationsmay, in some instances, be desired to inhibit the expression of aparticular B7 gene to an extent necessary to practice one of the methodsof the invention.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the oligonucleotide is administered in maintenance doses,ranging from 0.01 μg to 100 g per kg of body weight, once or more daily,to once every 20 years. In the case of in individual known or suspectedof being prone to an autoimmune or inflammatory condition, prophylacticeffects may be achieved by administration of preventative doses, rangingfrom 0.01 μg to 100 g per kg of body weight, once or more daily, to onceevery 20 years. In like fashion, an individual may be made lesssusceptible to an inflammatory condition that is expected to occur as aresult of some medical treatment, e.g., graft versus host diseaseresulting from the transplantation of cells, tissue or an organ into theindividual.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer or metered dose inhaler; intratracheal, intranasal, epidermaland transdermal, oral or parenteral. Parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular, administration. Oligonucleotides with at least one2′-O-methoxyethyl modification are believed to be particularly usefulfor oral administration.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Compositions for oraladministration also include pulsatile delivery compositions andbioadhesive composition as described in copending U.S. patentapplication Ser. No. 09/944,493, filed Aug. 22, 2001, and 09/935,316,filed Aug. 22, 2001, the entire disclosures of which are incorporatedherein by reference.

Compositions for parenteral, intrathecal or intraventricularadministration may include sterile aqueous solutions which may alsocontain buffers, diluents and other suitable additives.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years.

The following examples illustrate the invention and are not intended tolimit the same. Those skilled in the art will recognize, or be able toascertain through routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of the present invention.

The following examples are provided for illustrative purposes only andare not intended to limit the invention.

EXAMPLES Example 1 Synthesis of Nucleic Acids Oligonucleotides

Oligonucleotides were synthesized on an automated DNA synthesizer usingstandard phosphoramidite chemistry with oxidation using iodine.β-Cyanoethyldiisopropyl phosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of3H-1,2-benzodithiole-3-one-1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step.

The 2′-fluoro phosphorothioate oligonucleotides of the invention weresynthesized using 5′-dimethoxytrityl-3′-phosphoramidites and prepared asdisclosed in U.S. patent application Ser. No. 463,358, filed Jan. 11,1990, and Ser. No. 566,977, filed Aug. 13, 1990, which are assigned tothe same assignee as the instant application and which are incorporatedby reference herein. The 2′-fluoro oligonucleotides were prepared usingphosphoramidite chemistry and a slight modification of the standard DNAsynthesis protocol: deprotection was effected using methanolic ammoniaat room temperature.

The 2′-methoxy (2′-O-methyl)oligonucleotides of the invention weresynthesized using 2′-methoxy β-cyanoethyldiisopropyl-phosphoramidites(Chemgenes, Needham Mass.) and the standard cycle for unmodifiedoligonucleotides, except the wait step after pulse delivery of tetrazoleand base is increased to 360 seconds. Other 2′-alkoxy oligonucleotidesare synthesized by a modification of this method, using appropriate2′-modified amidites such as those available from Glen Research, Inc.,Sterling, Va. The 3′-base used to start the synthesis was a2′-deoxyribonucleotide. The 2′-O-propyl oligonucleotides of theinvention are prepared by a slight modification of this procedure.

The 2′ methoxyethoxy (2′-O—CH₂CH₂OCH₃) oligonucleotides of the inventionwere synthesized according to the method of Martin, Helv. Chim. Acta1995, 78, 486. For ease of synthesis, the last nucleotide was adeoxynucleotide. All 2′-O—CH₂CH₂OCH₃-cytosines were 5-methyl cytosines,which were synthesized according to the following procedures.

Synthesis of 5-Methyl Cytosine Monomers2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid which was crushed to a light tan powder (57 g, 85%crude yield). The material was used as is for further reactions.

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by tlc by first quenching the tlc sample with the addition ofMeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added to the later solutiondropwise, over a 45 minute period. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1 L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, tlc showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/Hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tlc showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2′-O-(Aminooxyethyl)nucleoside amidites and2′-O-(dimethylaminooxyethyl)nucleoside amidites2′-(Dimethylaminooxyethoxy)nucleoside amidites

2′-(Dimethylaminooxyethoxy)nucleoside amidites [also known in the art as2′-O-(dimethylaminooxyethyl)nucleoside amidites] are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O2-2′-anhydro-5-methyluridine

O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) weredissolved in dry pyridine (500 ml) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. Thereaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22,ethyl acetate) indicated a complete reaction. The solution wasconcentrated under reduced pressure to a thick oil. This was partitionedbetween dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L)and brine (1 L). The organic layer was dried over sodium sulfate andconcentrated under reduced pressure to a thick oil. The oil wasdissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) andthe solution was cooled to −10° C. The resulting crystalline product wascollected by filtration, washed with ethyl ether (3×200 mL) and dried(40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMRwere consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor was added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and withmanual stirring, ethylene glycol (350 mL, excess) was added cautiouslyat first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for are-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl)thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was stirred for 1 h. Solvent was removed undervacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridineas white foam (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O—[N,N-dimethylaminooxyethyl]-5

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na2SO4 and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) wasdried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P2O5 under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N1,N1-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa2SO4 and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy)nucleoside amidites

2′-(Aminooxyethoxy)nucleoside amidites [also known in the art as2′-O-(aminooxyethyl)nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2′-O-aminooxyethyl guanosine analog may be obtained by selective2′-O-alkylation of diaminopurine riboside. Multigram quantities ofdiaminopurine riboside may be purchased from Schering AG (Berlin) toprovide 2′-O-(2-ethylacetyl)diaminopurine riboside along with a minoramount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine ribosidemay be resolved and converted to 2′-O-(2-ethylacetyl)guanosine bytreatment with adenosine deaminase. (PCT WO94/02501). Standardprotection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE)nucleoside amidites

2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the artas 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, or2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleosideamidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowlyadded to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol)with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O2-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155 C for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine

To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO3solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH2Cl2:Et3N (20:1, v/v, with 1% triethylamine)gives the title compound.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) are added to a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Purification:

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides were purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Analytical gelelectrophoresis was accomplished in 20% acrylamide, 8 M urea, 45 mMTris-borate buffer, pH 7.0. Oligodeoxynucleotides and theirphosphorothioate analogs were judged from electrophoresis to be greaterthan 80% full length material.

B7 Antisense Oligonucleotides

A series of oligonucleotides with sequences designed to hybridize to thepublished human B7-1 (hB7-1) and murine B7-1 (mB7-1) mRNA sequences(Freeman et al., J. Immunol., 1989, 143, 2714, and Freeman et al., J.Exp. Med., 1991, 174, 625 respectively). The sequences of andmodifications to these oligonucleotides, and the location of each oftheir target sites on the hB7-1 mRNA, are given in Tables 1 and 2.Similarly, a series of oligonucleotides with sequences designed tohybridize to the human B7-2 (hB7-2) and murine B7-2 (mB7-2) mRNApublished sequences (respectively, Azuma et al., Nature, 1993, 366, 76;Chen et al., J. Immunol., 1994, 152, 4929) were synthesized. Thesequences of and modifications to these oligonucleotides and thelocation of each of their target sites on the hB7-2 mRNA are describedin Tables 3 and 4. Antisense oligonucleotides targeted to ICAM-1,including ISIS 2302 (SEQ ID NO: 17), have been described in U.S. Pat.No. 5,514,788, which issued May 7, 1996, hereby incorporated byreference. ISIS 1082 (SEQ ID NO: 102) and ISIS 3082 (SEQ ID NO: 101)have been previously described (Stepkowski et al., J. Immunol., 1994,153, 5336).

Subsequent to their initial cloning, alternative splicing events of B7transcripts have been reported. The reported alternative splicing forB7-1 is relatively simple, in that it results in messages extended 5′relative to the 5′ terminus of the human and murine B7-1 cDNA sequencesoriginally reported (Borriello et al., J. Immunol., 1994, 153, 5038;Inobe et al., J. Immunol., 1996, 157, 588). In order to retain thenumbering of the B7-1 sequences found in the references initiallyreporting B7-1 sequences, positions within these 5′ extensions of theinitially reported sequences have been given negative numbers (beginningwith position −1, the most 3′ base of the 5′ extension) in Tables 1 and2. The processing of murine B7-2 transcripts is considerably morecomplex than that so far reported for B7-1; for example, at least fivedistinct murine B7-2 mRNAs, and at least two distinct human B7-2 mRNAs,can be produced by alternative splicing events (Borriello et al., J.Immunol., 1995, 155, 5490; Freeman et al., WO 95/03408, published Feb.2, 1995; see also Jellis et al., Immunogenet., 1995, 42, 85). The natureof these splicing events is such that different 5′ exons are used toproduce distinct B7-2 mRNAs, each of which has a unique 5′ sequence butwhich share a 3′ portion consisting of some or all of the B7-2 sequenceinitially reported. As a result, positions within the 5′ extensions ofB7-2 messages cannot be uniquely related to a position within thesequence initially reported. Accordingly, in Table 3, a different set ofcoordinates (corresponding to those of SEQ ID NO: 1 of WO 95/03408) and,in Table 4, the exon number (as given in Borriello et al., J. Immunol.,1995, 155, 5490) is used to specify the location of targeted sequenceswhich are not included in the initially reported B7-2 sequence.Furthermore, although these 5′ extended messages contain potentialin-frame start codons upstream from the ones indicated in the initiallypublished sequences, for simplicity's sake, such additional potentialstart codons are not indicated in the description of target sites inTables 1-4.

In Tables 1-4, the following abbreviations are used: UTR, untranslatedregion; ORF, open reading frame; tIR, translation initiation region;tTR, translation termination region; FITC, fluorescein isothiocyanate.Chemical modifications are indicated as follows. Residues having 2′fluoro (2′F), 2′-methoxy (2′MO) or 2′-methoxyethoxy (2′ME) modificationare emboldened, with the type of modification being indicated by therespective abbreviations. Unless otherwise indicated, interresiduelinkages are phosphodiester linkages; phosphorothioate linkages areindicated by an “s” in the superscript position (e.g., T^(S)A). Targetpositions are numbered according to Freeman et al., J. Immunol., 1989,143:2714 (human B7-1 cDNA sequence; Table 1), Freeman et al., J. Exp.Med., 1991, 174, 625 (murine B7-1 cDNA sequence; Table 2), Azuma et al.,Nature, 1993, 366:76 (human B7-2 cDNA sequence; Table 3) and Chen etal., J. Immunol., 1994, 152:4929 (murine B7-2 cDNA sequence; Table 4).Nucleotide base codes are as given in 37 C.F.R. §1.822(b)(1).

TABLE 1 Sequences of Oligonucleotides Targeted to Human B7-1 mRNA SEQTarget Position; Site Oligonucleotide Sequence (5′->3′) ID ISIS #(and/or Description) and Chemical Modifications NO: 13797 0053-0072; 5′UTRG^(S)G^(S)G^(S)T^(S)A^(S)A^(S)G^(S)A^(S)C^(S)T^(S)C^(S)C^(S)A^(S)C^(S)T^(S)T^(S)C^(S)T^(S)G^(S)A22 13798 0132-0151; 5′ UTRG^(S)G^(S)G^(S)T^(S)C^(S)T^(S)C^(S)C^(S)A^(S)A^(S)A^(S)G^(S)G^(S)T^(S)T^(S)G^(S)T^(S)G^(S)G^(S)A23 13799 0138-0157; 5′ UTRG^(S)T^(S)T^(S)C^(S)C^(S)T^(S)G^(S)G^(S)G^(S)T^(S)C^(S)T^(S)G^(S)C^(S)A^(S)A^(S)A^(S)G^(S)G^(S)T24 13800 0158-0177; 5′ UTRA^(S)C^(S)A^(S)C^(S)A^(S)C^(S)A^(S)G^(S)A^(S)G^(S)A^(S)T^(S)T^(S)G^(S)G^(S)A^(S)G^(S)G^(S)G^(S)T25 13801 0193-0212; 5′ UTRG^(S)C^(S)T^(S)C^(S)A^(S)C^(S)G^(S)T^(S)A^(S)G^(S)A^(S)A^(S)G^(S)A^(S)C^(S)C^(S)C^(S)T^(S)C^(S)C26 13802 0217-0236; 5′ UTRG^(S)G^(S)C^(S)A^(S)G^(S)G^(S)G^(S)C^(S)T^(S)G^(S)A^(S)T^(S)G^(S)A^(S)C^(S)A^(S)A^(S)T^(S)C^(S)C27 13803 0226-0245; 5′ UTRT^(S)G^(S)C^(S)A^(S)A^(S)A^(S)A^(S)C^(S)A^(S)G^(S)G^(S)C^(S)A^(S)G^(S)G^(S)G^(S)C^(S)T^(S)G^(S)A28 13804 0246-0265; 5′ UTRA^(S)G^(S)A^(S)C^(S)G^(S)A^(S)G^(S)G^(S)G^(S)C^(S)A^(S)C^(S)T^(S)T^(S)C^(S)C^(S)C^(S)A^(S)G^(S)G29 13805 0320-0339; tIRC^(S)C^(S)T^(S)G^(S)C^(S)C^(S)T^(S)C^(S)C^(S)G^(S)T^(S)G^(S)T^(S)G^(S)T^(S)G^(S)G^(S)C^(S)C^(S)C30 13806 0380-0399; 5′ ORFG^(S)A^(S)C^(S)C^(S)A^(S)G^(S)C^(S)C^(S)A^(S)G^(S)C^(S)A^(S)C^(S)C^(S)A^(S)A^(S)G^(S)A^(S)G^(S)C31 13807 0450-0469; 5′ ORFC^(S)C^(S)A^(S)C^(S)A^(S)G^(S)G^(S)A^(S)C^(S)A^(S)G^(S)C^(S)G^(S)T^(S)T^(S)G^(S)C^(S)C^(S)A^(S)C32 13808 0568-0587; 5′ ORFC^(S)C^(S)G^(S)G^(S)T^(S)T^(S)C^(S)T^(S)T^(S)G^(S)T^(S)A^(S)C^(S)T^(S)C^(S)G^(S)G^(S)G^(S)C^(S)C33 13809 0634-0653; central ORFG^(S)C^(S)C^(S)C^(S)T^(S)C^(S)G^(S)T^(S)C^(S)A^(S)G^(S)A^(S)T^(S)G^(S)G^(S)G^(S)C^(S)G^(S)C^(S)A51 13810 0829-0848; central ORFC^(S)C^(S)A^(S)A^(S)C^(S)C^(S)A^(S)G^(S)G^(S)A^(S)G^(S)A^(S)G^(S)G^(S)T^(S)G^(S)A^(S)G^(S)G^(S)C34 13811 1102-1121; 3′ ORFG^(S)G^(S)C^(S)A^(S)A^(S)A^(S)G^(S)C^(S)A^(S)G^(S)T^(S)A^(S)G^(S)G^(S)T^(S)C^(S)A^(S)G^(S)G^(S)C35 13812 1254-1273; 3′-UTRG^(S)C^(S)C^(S)T^(S)C^(S)A^(S)T^(S)G^(S)A^(S)T^(S)C^(S)C^(S)C^(S)C^(S)A^(S)C^(S)G^(S)A^(S)T^(S)C36 13872 (scrambled # 13812)A^(S)G^(S)T^(S)C^(S)C^(S)T^(S)A^(S)C^(S)T^(S)A^(S)C^(S)C^(S)A^(S)G^(S)C^(S)C^(S)G^(S)C^(S)C^(S)T52 12361 0056-0075; 5′ UTRT^(S)C^(S)A^(S)G^(S)G^(S)G^(S)T^(S)A^(S)A^(S)G^(S)A^(S)C^(S)T^(S)C^(S)C^(S)A^(S)C^(S)T^(S)T^(S)C38 12348 0056-0075; 5′ UTR T C A G G G^(S)T^(S)A^(S)A^(S)G^(S)A^(S)C^(S)T^(S) C ^(S) C A C T T C 38 (2′ME)12473 0056-0075; 5′ UTR T^(S) C ^(S) A ^(S) G ^(S) G ^(S) G^(S)T^(S)A^(S)A^(S)G^(S)A^(S)C^(S)T^(S)C^(S) C ^(S) A ^(S) C ^(S) T ^(S)T ^(S) C 38 (2′FI) 12362 0143-0162; 5′ UTRA^(S)G^(S)G^(S)G^(S)T^(S)G^(S)T^(S)T^(S)C^(S)C^(S)T^(S)G^(S)G^(S)G^(S)T^(S)C^(S)T^(S)C^(S)C^(S)A39 12349 0143-0162; 5′ UTR A G G G T G^(S)T^(S)T^(S)C^(S)C^(S)T^(S)G^(S)G^(S) G ^(S) T C T C C A 39 (2′ME)12474 0143-0162; 5′ UTR A ^(S) G ^(S) G ^(S) G ^(S) T ^(S) G^(S)T^(S)T^(S)C^(S)C^(S)T^(S)G^(S)G^(S)G^(S) T ^(S) C ^(S) T ^(S) C ^(S)C ^(S) A 39 (2′F1) 12363 0315-0334; tIRC^(S)T^(S)C^(S)C^(S)G^(S)T^(S)G^(S)T^(S)G^(S)T^(S)G^(S)G^(S)C^(S)C^(S)C^(S)A^(S)T^(S)G^(S)G^(S)C40 12350 0315-0334; tIR C T C C G T^(S)G^(S)T^(S)G^(S)T^(S)G^(S)G^(S)C^(S) C C A T G G C 40 (2′ME) 124750315-0334; tIR C ^(S) T ^(S) C ^(S) C ^(S) G ^(S) T^(S)G^(S)T^(S)G^(S)T^(S)G^(S)G^(S)C^(S)C^(S) C ^(S) A ^(S) T ^(S) G ^(S)G ^(S) C 40 (2′FI) 12364 0334-0353; 5′ ORFG^(S)G^(S)A^(S)T^(S)G^(S)G^(S)T^(S)G^(S)A^(S)T^(S)G^(S)T^(S)T^(S)C^(S)C^(S)C^(S)T^(S)G^(S)C^(S)C41 12351 0334-0353; 5′ ORF G G A T G G^(S)T^(S)G^(S)A^(S)T^(S)G^(S)T^(S)T^(S) C C C T G C C 41 (2′ME) 124760334-0353; 5′ ORF G ^(S) G ^(S) A ^(S) T ^(S) G ^(S) G^(S)T^(S)G^(S)A^(S)T^(S)G^(S)T^(S)T^(S)C^(S) C ^(S) C ^(S) T ^(S) G ^(S)C ^(S) C 41 (2′FI) 12365 0387-0406; 5′ ORFT^(S)G^(S)A^(S)G^(S)A^(S)A^(S)A^(S)G^(S)A^(S)C^(S)C^(S)A^(S)G^(S)C^(S)C^(S)A^(S)G^(S)C^(S)A^(S)C42 12352 0387-0406; 5′ ORF T G A G A A^(S)A^(S)G^(S)A^(S)C^(S)C^(S)A^(S)G^(S) C ^(S) C A G C A C 42 (2′ME)12477 0387-0406; 5′ ORF T ^(S) G ^(S) A ^(S) G ^(S) A ^(S) A^(S)A^(S)G^(S)A^(S)C^(S)C^(S)A^(S)G^(S)C^(S) C ^(S) A ^(S) G ^(S) C ^(S)A ^(S) C 42 (2′FI) 12366 0621-0640; central ORFG^(S)G^(S)G^(S)C^(S)G^(S)C^(S)A^(S)G^(S)A^(S)G^(S)C^(S)C^(S)A^(S)G^(S)G^(S)A^(S)T^(S)C^(S)A^(S)C43 12353 0621-0640; central ORF G G G C G C^(S)A^(S)G^(S)A^(S)G^(S)C^(S)C^(S)A^(S) G G A T C A C 43 (2′ME) 124780621-0640; central ORF G ^(S) G ^(S) G ^(S) C ^(S) G ^(S) C^(S)A^(S)G^(S)A^(S)G^(S)C^(S)C^(S)A^(S)G^(S) G ^(S) A ^(S) T ^(S) C ^(S)A ^(S) C 43 (2′FI) 12367 1042-1061; 3′ ORFG^(S)G^(S)C^(S)C^(S)C^(S)A^(S)G^(S)G^(S)A^(S)T^(S)G^(S)G^(S)G^(S)A^(S)G^(S)C^(S)A^(S)G^(S)G^(S)T44 12354 1042-1061; 3′ ORF G G C C C A^(S)G^(S)G^(S)A^(S)T^(S)G^(S)G^(S)G^(S) A G C A G G T 44 (2′ME) 124791042-1061; 3′ ORF G ^(S) G ^(S) C ^(S) C ^(S) C ^(S) A^(S)G^(S)G^(S)A^(S)T^(S)G^(S)G^(S)G^(S)A^(S) G ^(S) C ^(S) A ^(S) G ^(S)G ^(S) T 44 (2′FI) 12368 1069-1088; tTRA^(S)G^(S)G^(S)G^(S)C^(S)G^(S)T^(S)A^(S)C^(S)A^(S)C^(S)T^(S)T^(S)T^(S)C^(S)C^(S)C^(S)T^(S)T^(S)C45 12355 1069-1088; tTR A G G G C G^(S)T^(S)A^(S)C^(S)A^(S)C^(S)T^(S)T^(S) T C C C T T C 45 (2′ME) 124801069-1088; tTR A ^(S) G ^(S) G ^(S) G ^(S) C ^(S) G^(S)T^(S)A^(S)C^(S)A^(S)C^(S)T^(S)T^(S)T^(S) C ^(S) C ^(S) C ^(S) T ^(S)T ^(S) C 45 (2′FI) 12369 1100-1209; tTRC^(S)A^(S)G^(S)C^(S)C^(S)C^(S)C^(S)T^(S)T^(S)G^(S)C^(S)T^(S)T^(S)C^(S)T^(S)G^(S)C^(S)G^(S)G^(S)A46 12356 1100-1209; tTR C A G C C C^(S)C^(S)T^(S)T^(S)G^(S)C^(S)T^(S)T^(S)C^(S) T G C G G A 46 (2′ME) 124811100-1209; tTR C ^(S) A ^(S) G ^(S) C ^(S) C ^(S) C^(S)C^(S)T^(S)T^(S)G^(S)C^(S)T^(S)T^(S)C^(S) T ^(S) G ^(S) C ^(S) G ^(S)G ^(S) A 46 (2′FI) 12370 1360-1380; 3′ UTRA^(S)A^(S)G^(S)G^(S)A^(S)G^(S)A^(S)G^(S)G^(S)G^(S)A^(S)T^(S)G^(S)C^(S)C^(S)A^(S)G^(S)C^(S)C^(S)A47 12357 1360-1380; 3′ UTR A A G G A G^(S)A^(S)G^(S)G^(S)G^(S)A^(S)T^(S)G^(S) C C A G C C A 47 (2′ME) 124821360-1380; 3′ UTR A ^(S) A ^(S) G ^(S) G ^(S) A ^(S) G^(S)A^(S)G^(S)G^(S)G^(S)A^(S)T^(S)G^(S)C^(S) C ^(S) A ^(S) G ^(S) C ^(S)C ^(S) A 47 (2′FI) 12914 (-0038 to -0059; C ^(S) T ^(S) G ^(S) T ^(S) T^(S) A ^(S) C ^(S) T ^(S) T ^(S) T ^(S) A ^(S) C ^(S) A ^(S) G ^(S) A^(S) G ^(S) G ^(S) G ^(S) T ^(S) T 48 5′ UTR of ^(S) T ^(S) G (2′MO)alternative mRNA) 12915 (-0035 to -0059; C ^(S) T ^(S) T ^(S) C ^(S) T^(S) G ^(S) T ^(S) T ^(S) A ^(S) C ^(S) T ^(S) T ^(S) T ^(S) A ^(S) C^(S) A ^(S) G ^(S) A ^(S) G ^(S) G 49 5′ UTR of ^(S) G ^(S) T ^(S) T^(S) T ^(S) G (2′MO) alternative mRNA) 13498 (-0038 to -0058; C ^(S) T^(S) G ^(S) T ^(S) T ^(S) A ^(S) C ^(S) T ^(S) T ^(S) T ^(S) A ^(S) C^(S) A ^(S) G ^(S) A ^(S) G ^(S) G ^(S) G ^(S) T ^(S) T 50 5′ UTR of^(S) T(2′ME) alternative mRNA) 13499 (-0038 to -0058;C T G T T A C T T T A C A G A G G G T T T 50 5′ UTR of (2′ME)alternative mRNA)

TABLE 2 Sequences of Oligonucleotides Targeted to Murine B7-1 mRNA SEQOligonucleotide Sequence (5′->3′) ID ISIS # Target Position; SiteChemical Modifications NO: 14419 0009-0028; 5′ UTRA^(S)G^(S)T^(S)A^(S)A^(S)G^(S)A^(S)G^(S)T^(S)C^(S)T^(S)A^(S)T^(S)T^(S)G^(S)A^(S)G^(S)G^(S)T^(S)A53 14420 0041-0060; 5′ UTRG^(S)G^(S)T^(S)T^(S)G^(S)A^(S)G^(S)T^(S)T^(S)T^(S)C^(S)A^(S)C^(S)A^(S)A^(S)C^(S)C^(S)T^(S)G^(S)A54 14421 0071-0091; 5′ UTRG^(S)T^(S)C^(S)C^(S)A^(S)G^(S)A^(S)G^(S)A^(S)A^(S)T^(S)G^(S)G^(S)A^(S)A^(S)C^(S)A^(S)G^(S)A^(S)G55 14422 0109-0128; 5′ UTRG^(S)G^(S)C^(S)A^(S)T^(S)C^(S)C^(S)A^(S)C^(S)C^(S)C^(S)G^(S)G^(S)C^(S)A^(S)G^(S)A^(S)T^(S)G^(S)C56 14423 0114-0133; 5′ UTRT^(S)G^(S)G^(S)A^(S)T^(S)G^(S)G^(S)C^(S)A^(S)T^(S)C^(S)C^(S)A^(S)C^(S)C^(S)C^(S)G^(S)G^(S)C^(S)A57 14424 0168-0187; 5′ UTRA^(S)G^(S)G^(S)C^(S)A^(S)C^(S)C^(S)T^(S)C^(S)C^(S)T^(S)A^(S)G^(S)G^(S)C^(S)T^(S)C^(S)A^(S)C^(S)A58 14425 0181-0200; 5′ UTRG^(S)C^(S)C^(S)A^(S)A^(S)T^(S)G^(S)G^(S)A^(S)G^(S)C^(S)T^(S)T^(S)A^(S)G^(S)G^(S)C^(S)A^(S)C^(S)C59 14426 0208-0217; 5′ UTRC^(S)A^(S)T^(S)G^(S)A^(S)T^(S)G^(S)G^(S)G^(S)G^(S)A^(S)A^(S)A^(S)G^(S)C^(S)C^(S)A^(S)G^(S)G^(S)A60 14427 0242-0261; tIRA^(S)A^(S)T^(S)T^(S)G^(S)C^(S)A^(S)A^(S)G^(S)C^(S)C^(S)A^(S)T^(S)A^(S)G^(S)C^(S)T^(S)T^(S)C^(S)A61 14428 0393-0412; 5′ ORFC^(S)G^(S)G^(S)C^(S)A^(S)A^(S)G^(S)G^(S)C^(S)A^(S)G^(S)C^(S)A^(S)A^(S)T^(S)A^(S)C^(S)C^(S)T^(S)T62 14909 0478-0497; 5′ ORFC^(S)C^(S)C^(S)A^(S)G^(S)C^(S)A^(S)A^(S)T^(S)G^(S)A^(S)C^(S)A^(S)G^(S)A^(S)C^(S)A^(S)G^(S)C^(S)A63 14910 0569-0588; central ORFG^(S)G^(S)T^(S)C^(S)T^(S)G^(S)A^(S)A^(S)A^(S)G^(S)G^(S)A^(S)C^(S)C^(S)A^(S)G^(S)G^(S)C^(S)C^(S)C64 14911 0745-0764; central ORFT^(S)G^(S)G^(S)G^(S)A^(S)A^(S)A^(S)C^(S)C^(S)C^(S)C^(S)C^(S)G^(S)G^(S)A^(S)A^(S)G^(S)C^(S)A^(S)A65 14912 0750-0769; central ORFG^(S)G^(S)C^(S)T^(S)T^(S)T^(S)G^(S)G^(S)G^(S)A^(S)A^(S)A^(S)C^(S)C^(S)C^(S)C^(S)C^(S)G^(S)G^(S)A66 14913 0825-0844; 3′ ORFT^(S)C^(S)A^(S)G^(S)A^(S)T^(S)T^(S)C^(S)A^(S)G^(S)G^(S)A^(S)T^(S)C^(S)C^(S)T^(S)G^(S)G^(S)G^(S)A67 14914 0932-0951; 3′ ORFC^(S)C^(S)C^(S)A^(S)G^(S)G^(S)T^(S)G^(S)A^(S)A^(S)G^(S)T^(S)C^(S)C^(S)T^(S)C^(S)T^(S)G^(S)A^(S)C68 14915 1001-1020; 3′ ORFC^(S)T^(S)G^(S)C^(S)G^(S)C^(S)C^(S)G^(S)A^(S)A^(S)T^(S)C^(S)C^(S)T^(S)G^(S)C^(S)C^(S)C^(S)C^(S)A69 14916 1125-1144; tTRC^(S)A^(S)G^(S)G^(S)C^(S)C^(S)C^(S)G^(S)A^(S)A^(S)G^(S)G^(S)T^(S)A^(S)A^(S)G^(S)G^(S)C^(S)T^(S)G70 14917 1229-1248; 3′ UTRT^(S)C^(S)A^(S)G^(S)C^(S)T^(S)A^(S)G^(S)C^(S)A^(S)C^(S)G^(S)G^(S)T^(S)G^(S)C^(S)T^(S)G^(S)A^(S)A71 14918 1329-1348; 3′ UTRG^(S)G^(S)C^(S)C^(S)C^(S)A^(S)G^(S)C^(S)A^(S)A^(S)A^(S)G^(S)T^(S)T^(S)G^(S)C^(S)C^(S)C^(S)G^(S)T72 14919 1377-1393; 3′ UTRC^(S)C^(S)A^(S)C^(S)C^(S)A^(S)C^(S)A^(S)G^(S)T^(S)G^(S)G^(S)G^(S)C^(S)T^(S)C^(S)A^(S)G^(S)C^(S)C73 12912 -0067 to -0049; 5′ UTR G ^(S) G ^(S) C ^(S) C ^(S) A ^(S) T^(S) G ^(S) A ^(S) G ^(S) G ^(S) G ^(S) C ^(S) A ^(S) A ^(S) T ^(S) C^(S) T ^(S) A ^(S) A 74 (2′MO) 12913 -0067 to -0047; 5′ UTR G ^(S) T^(S) G ^(S) G ^(S) C ^(S) C ^(S) A ^(S) T ^(S) G ^(S) A ^(S) G ^(S) G^(S) G ^(S) C ^(S) A ^(S) A ^(S) T ^(S) C ^(S) T ^(S) A ^(S) A 75 (2′MO)13496 -0067 to -0047; 5′ UTR G ^(S) T ^(S) G ^(S) G ^(S) C ^(S) C ^(S) A^(S) T ^(S) G ^(S) A ^(S) G ^(S) G ^(S) G ^(S) C ^(S) A ^(S) A ^(S) T^(S) C ^(S) T ^(S) A ^(S) A 75 (2′ME) 13497 -0067 to -0047; 5′ UTRG T G G C C A T G A G G G C A A T C T A A 75 (2′ME)

TABLE 3 Sequences of Oligonucleotides Targeted to Human B7-2 mRNA SEQ IDISIS # Target Position*; Site** Oligonucleotide Sequence (5′->3′) NO:9133 1367-1386; 3′-UTRT^(S)T^(S)C^(S)C^(S)A^(S)G^(S)G^(S)T^(S)C^(S)A^(S)T^(S)G^(S)A^(S)G^(S)C^(S)C^(S)A^(S)T^(S)T^(S)A3 10715 scrambled control of # 9133G^(S)A^(S)T^(S)T^(S)T^(S)A^(S)A^(S)C^(S)A^(S)T^(S)T^(S)T^(S)G^(S)G^(S)C^(S)G^(S)C^(S)C^(S)C^(S)A76 9134 1333-1352; 3′-UTRC^(S)A^(S)T^(S)A^(S)A^(S)G^(S)G^(S)T^(S)G^(S)T^(S)G^(S)C^(S)T^(S)C^(S)T^(S)G^(S)A^(S)A^(S)G^(S)T^(S)G4 9135 1211-1230; 3′-UTRT^(S)T^(S)A^(S)C^(S)T^(S)C^(S)A^(S)T^(S)G^(S)G^(S)T^(S)A^(S)A^(S)T^(S)G^(S)T^(S)C^(S)T^(S)T^(S)T^(S)5 9136 1101-1120; tTRA^(S)T^(S)T^(S)A^(S)A^(S)A^(S)A^(S)A^(S)C^(S)A^(S)T^(S)G^(S)T^(S)A^(S)T^(S)C^(S)A^(S)C^(S)T^(S)T^(S)6 10716 (scrambled # 9136)A^(S)A^(S)A^(S)G^(S)T^(S)T^(S)A^(S)C^(S)A^(S)A^(S)C^(S)A^(S)T^(S)T^(S)A^(S)T^(S)A^(S)T^(S)C^(S)T77 9137 0054-0074; 5′-UTRG^(S)G^(S)A^(S)A^(S)C^(S)A^(S)C^(S)A^(S)G^(S)A^(S)A^(S)G^(S)C^(S)A^(S)A^(S)G^(S)G^(S)T^(S)G^(S)G^(S)T7 9138 0001-0020; 5′-UTRC^(S)C^(S)G^(S)T^(S)A^(S)C^(S)C^(S)T^(S)C^(S)C^(S)T^(S)A^(S)A^(S)G^(S)G^(S)C^(S)T^(S)C^(S)C^(S)T8 9139 0133-0152; tIRC^(S)C^(S)C^(S)A^(S)T^(S)A^(S)G^(S)T^(S)G^(S)C^(S)T^(S)G^(S)T^(S)C^(S)A^(S)C^(S)A^(S)A^(S)A^(S)T9 10877 (scrambled # 9139)A^(S)G^(S)T^(S)G^(S)C^(S)G^(S)A^(S)T^(S)T^(S)C^(S)T^(S)C^(S)A^(S)A^(S)A^(S)C^(S)C^(S)T^(S)A^(S)C78 10367 0073-0092; 5′-UTRG^(S)C^(S)A^(S)C^(S)A^(S)G^(S)C^(S)A^(S)G^(S)C^(S)A^(S)T^(S)T^(S)C^(S)C^(S)C^(S)A^(S)A^(S)G^(S)G10 10368 0240-0259; 5′ ORFT^(S)T^(S)G^(S)C^(S)A^(S)A^(S)A^(S)T^(S)T^(S)G^(S)G^(S)C^(S)A^(S)T^(S)G^(S)G^(S)C^(S)A^(S)G^(S)G11 10369 1122-1141; 3′-UTRT^(S)G^(S)G^(S)T^(S)A^(S)T^(S)G^(S)G^(S)G^(S)C^(S)T^(S)T^(S)T^(S)A^(S)C^(S)T^(S)C^(S)T^(S)T^(S)T12 10370 1171-1190; 3′-UTRA^(S)A^(S)A^(S)A^(S)G^(S)G^(S)T^(S)T^(S)G^(S)C^(S)C^(S)C^(S)A^(S)G^(S)G^(S)A^(S)A^(S)C^(S)G^(S)G13 10371 1233-1252; 3′-UTRG^(S)G^(S)G^(S)A^(S)G^(S)T^(S)C^(S)C^(S)T^(S)G^(S)G^(S)A^(S)G^(S)C^(S)C^(S)C^(S)C^(S)C^(S)T^(S)T14 10372 1353-1372; 3′-UTRC^(S)C^(S)A^(S)T^(S)T^(S)A^(S)A^(S)G^(S)C^(S)T^(S)G^(S)G^(S)G^(S)C^(S)T^(S)T^(S)G^(S)G^(S)C^(S)C15 11149 0019-0034; 5′-UTRT^(S)A^(S)T^(S)T^(S)T^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C79 11151 0020-0034; 5′-UTRT^(S)A^(S)T^(S)T^(S)T^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C80 11150 0021-0034; 5′-UTRT^(S)A^(S)T^(S)T^(S)T^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C 8110373 0011-0030; 5′-UTRT^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C^(S)C^(S)T^(S)C^(S)G16 10721 (scrambled # 10373)C^(S)G^(S)A^(S)C^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)T^(S)G^(S)C^(S)G^(S)C^(S)T^(S)C^(S)C^(S)T^(S)C82 10729 (5′FITC # 10373)T^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C^(S)C^(S)T^(S)C^(S)C16 10782 (5′cholesterol # 10373)T^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C^(S)C^(S)T^(S)C^(S)C16 # 10373 Deletion Derivatives: 10373 0011-0030; 5′-UTRT^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C^(S)C^(S)T^(S)C^(S)C16 10888 0011-0026; 5′-UTRA^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C^(S)C^(S)T^(S)C^(S)C83 10889 0015-0030; 5′-UTRT^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C84 10991 0015-0024; 5′-UTRC^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C 85 109920015-0025; 5′-UTR G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C 8610993 0015-0026; 5′-UTRA^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C 87 109940015-0027; 5′-UTRG^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C 88 109950015-0028; 5′-UTRC^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C 8910996 0015-0029; 5′-UTRG^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C90 11232 0017-0029; 5′ UTRG^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T 91 #10996 Derivatives: 10996 0015-0029; 5′-UTRG^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C90 11806 (scrambled # 10996)G^(S)C^(S)C^(S)G^(S)C^(S)C^(S)G^(S)C^(S)C^(S)A^(S)A^(S)G^(S)T^(S)C^(S)T92 11539 (fully 2′MO # 10996) G ^(S) C ^(S) G ^(S) A ^(S) G ^(S) C ^(S)T ^(S) C ^(S) C ^(S) C ^(S) C ^(S) G ^(S) T ^(S) A ^(S)C (2′MO) 90 11540(control for # 11539) G ^(S) C ^(S) C ^(S) G ^(S) C ^(S) C ^(S) G ^(S) C^(S) C ^(S) A ^(S) A ^(S) G ^(S) T ^(S) C ^(S)T (2′MO) 92 11541 (#10996 7-base “gapmer”) G ^(S) C ^(S) G ^(S) A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S) G ^(S) T ^(S) A ^(S) C (2′MO) 9011542 (control for # 11541) G ^(S) C ^(S) C ^(S) G^(S)C^(S)C^(S)G^(S)C^(S)C^(S)A^(S)A^(S) G ^(S) T ^(S) C ^(S) T (2′MO) 9211543 (# 10996 9-base “gapmer”) G ^(S) C ^(S) G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S) T ^(S) A ^(S) C (2′MO)90 11544 (control for # 11543) G ^(S) C ^(S) C^(S)G^(S)C^(S)C^(S)G^(S)C^(S)C^(S)A^(S)A^(S)G^(S) T ^(S) C ^(S) T (2′MO)92 11545 (# 10996 5′ “wingmer”) G ^(S) C ^(S) G ^(S) A ^(S) G ^(S) C^(S) T ^(S) C ^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C (2′MO) 90 11546(control for # 11545) G ^(S) C ^(S) C ^(S) G ^(S) C ^(S) C ^(S) G ^(S) C^(S)C^(S)A^(S)A^(S)G^(S)T^(S)C^(S)T (2′MO) 92 11547 (# 10996 3′“wingmer”) G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S) C ^(S) C ^(S) C ^(S) C^(S) G ^(S) T ^(S) A ^(S) C (2′MO) 90 11548 (control for # 11547)G^(S)G^(S)C^(S)G^(S)C^(S)C^(S)G^(S) C ^(S) C ^(S) A ^(S) A ^(S) G ^(S) T^(S) C ^(S) T (2′MO) 92 12496 ((2′-5′)A₄ # 10996) GCGAGCTCCCCGTAC 9013107 ((2′-5′)A₄ # 10996)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C90 12492 ((2′-5′)A₄ # 10996) G ^(S) C ^(S) G ^(S) A ^(S) G ^(S) C ^(S) T^(S) C ^(S) C ^(S) C ^(S) C ^(S) G ^(S) T ^(S) A ^(S) C (2′MO) 90 12495((2′-5′)A₄ # 10996) G ^(S) C ^(S) G ^(S) A ^(S) G ^(S) C ^(S) T ^(S) C^(S) C ^(S) C ^(S) C ^(S) G ^(S) T ^(S) A ^(S) C (2′MO) 90 12887(1-24 of SEQ ID NO:1 of WO G ^(S) A ^(S) G ^(S) A ^(S) A ^(S) G ^(S) C^(S) A ^(S) A ^(S) A ^(S) G ^(S) C ^(S) T ^(S) T ^(S) T ^(S) C ^(S) A^(S) C ^(S) C ^(S) C- 93 95/03408; alternative mRNA) ^(S) T ^(S) G ^(S)T ^(S) G (2′MO) 12888 (1-22 of SEQ ID NO:1 of WO G ^(S) A ^(S) A ^(S) G^(S) C ^(S) A ^(S) A ^(S) A ^(S) G ^(S) C ^(S) T ^(S) T ^(S) T ^(S) C^(S) A ^(S) C ^(S) C ^(S) C ^(S) T ^(S) G ^(S) T ^(S) 9495/03408; alternative mRNA) G (2′MO) 12889 (1-19 of SEQ ID NO:1 of WO G^(S) C ^(S) A ^(S) A ^(S) A ^(S) G ^(S) C ^(S) T ^(S) T ^(S) T ^(S) C^(S) A ^(S) C ^(S) C ^(S) C ^(S) T ^(S) G ^(S) T ^(S) G 9595/03408; alternative mRNA) (2′MO) 12890 0001-0024 C ^(S) T ^(S) C ^(S)C ^(S) C ^(S) C ^(S) G ^(S) T ^(S) A ^(S) C ^(S) C ^(S) T ^(S) C ^(S) C^(S) T ^(S) A ^(S) A ^(S) G ^(S) G ^(S) C- 96 ^(S) T ^(S) C ^(S) C ^(S)T (2′MO) 12891 0001-0022 C ^(S) C ^(S) C ^(S) C ^(S) G ^(S) T ^(S) A^(S) C ^(S) C ^(S) T ^(S) C ^(S) C ^(S) T ^(S) A ^(S) A ^(S) G ^(S) G^(S) C ^(S) T ^(S) C ^(S) C ^(S) 97 T (2′MO) 12892 0001-0020 C ^(S) C^(S) G ^(S) T ^(S) A ^(S) C ^(S) C ^(S) T ^(S) C ^(S) C ^(S) T ^(S) A^(S) A ^(S) G ^(S) G ^(S) C ^(S) T ^(S) C ^(S) C 98 (2′MO)

TABLE 4 Sequences of Oligonucleotides Targeted to Murine B7-2 mRNA SEQID ISIS # Target Position; Site Oligonucleotide Sequence (5′->3′) NO:11347 1094-1113; 3′ UTRA^(S)G^(S)A^(S)A^(S)T^(S)T^(S)C^(S)C^(S)A^(S)A^(S)T^(S)C^(S)A^(S)G^(S)C^(S)T^(S)G^(S)A^(S)G^(S)A121 11348 1062-1081; 3′ UTRT^(S)C^(S)T^(S)G^(S)A^(S)G^(S)A^(S)A^(S)A^(S)C^(S)T^(S)C^(S)T^(S)G^(S)C^(S)A^(S)C^(S)T^(S)T^(S)C122 11349 1012-1031; 3′ UTRT^(S)C^(S)C^(S)T^(S)C^(S)A^(S)G^(S)G^(S)C^(S)T^(S)C^(S)T^(S)C^(S)A^(S)C^(S)T^(S)G^(S)C^(S)C^(S)T123 11350 0019-1138; 5′ UTRG^(S)G^(S)T^(S)T^(S)G^(S)T^(S)T^(S)C^(S)A^(S)A^(S)G^(S)T^(S)C^(S)C^(S)G^(S)T^(S)G^(S)C^(S)T^(S)G124 11351 0037-0056; 5′ UTRA^(S)C^(S)A^(S)C^(S)G^(S)T^(S)C^(S)T^(S)A^(S)C^(S)A^(S)G^(S)G^(S)A^(S)G^(S)T^(S)C^(S)T^(S)G^(S)G103 11352 0089-0108; tIRC^(S)A^(S)A^(S)G^(S)C^(S)C^(S)C^(S)A^(S)T^(S)G^(S)G^(S)T^(S)G^(S)C^(S)A^(S)T^(S)C^(S)T^(S)G^(S)G104 11353 0073-0092; tIRC^(S)T^(S)G^(S)G^(S)G^(S)G^(S)T^(S)C^(S)C^(S)A^(S)T^(S)C^(S)G^(S)T^(S)G^(S)G^(S)G^(S)T^(S)G^(S)C105 11354 0007-0026; 5′ UTRC^(S)C^(S)G^(S)T^(S)G^(S)C^(S)T^(S)G^(S)C^(S)C^(S)T^(S)A^(S)C^(S)A^(S)G^(S)G^(S)A^(S)G^(S)C^(S)C106 11695 0058-0077; 5′ UTRG^(S)G^(S)T^(S)G^(S)C^(S)T^(S)T^(S)C^(S)C^(S)G^(S)T^(S)A^(S)A^(S)G^(S)T^(S)T^(S)C^(S)T^(S)G^(S)G107 11696 0096-0117; tIRG^(S)G^(S)A^(S)T^(S)T^(S)G^(S)C^(S)C^(S)A^(S)A^(S)G^(S)C^(S)C^(S)C^(S)A^(S)T^(S)G^(S)G^(S)T^(S)G108 11866 (scrambled # 11696)C^(S)T^(S)A^(S)A^(S)G^(S)T^(S)A^(S)G^(S)T^(S)G^(S)C^(S)T^(S)A^(S)G^(S)C^(S)C^(S)G^(S)G^(S)G^(S)A109 11697 0148-0167; 5′ ORFT^(S)G^(S)C^(S)G^(S)T^(S)C^(S)T^(S)C^(S)C^(S)A^(S)C^(S)G^(S)G^(S)A^(S)A^(S)A^(S)C^(S)A^(S)G^(S)C110 11698 0319-0338; 5′ ORFG^(S)T^(S)G^(S)C^(S)G^(S)G^(S)C^(S)C^(S)C^(S)A^(S)G^(S)G^(S)T^(S)A^(S)C^(S)T^(S)T^(S)G^(S)G^(S)C111 11699 0832-0851; 3′ ORFA^(S)C^(S)A^(S)A^(S)G^(S)G^(S)A^(S)G^(S)G^(S)A^(S)G^(S)G^(S)G^(S)C^(S)C^(S)A^(S)C^(S)A^(S)G^(S)T112 11700 0753-0772; 3′ ORFT^(S)G^(S)A^(S)G^(S)A^(S)G^(S)G^(S)T^(S)T^(S)T^(S)G^(S)G^(S)A^(S)G^(S)G^(S)A^(S)A^(S)A^(S)T^(S)C113 11701 0938-0957; 3′ ORFG^(S)A^(S)T^(S)A^(S)G^(S)T^(S)C^(S)T^(S)C^(S)T^(S)C^(S)T^(S)G^(S)T^(S)C^(S)A^(S)G^(S)C^(S)G^(S)T114 11702 0890-0909; 3′ ORFG^(S)T^(S)T^(S)G^(S)C^(S)T^(S)G^(S)G^(S)G^(S)C^(S)C^(S)T^(S)G^(S)C^(S)T^(S)A^(S)G^(S)G^(S)C^(S)T115 11865 (scrambled # 11702)C^(S)T^(S)A^(S)G^(S)G^(S)T^(S)C^(S)T^(S)C^(S)G^(S)T^(S)C^(S)G^(S)T^(S)C^(S)G^(S)G^(S)T^(S)G^(S)G116 11703 1003-1022; tTRT^(S)C^(S)T^(S)C^(S)A^(S)C^(S)T^(S)G^(S)C^(S)C^(S)T^(S)T^(S)C^(S)A^(S)C^(S)T^(S)C^(S)T^(S)G^(S)C117 13100 Exon 1 (Borriello et al., G ^(S) T ^(S) A ^(S) C ^(S) C ^(S) A^(S) G ^(S) A ^(S) T ^(S) G ^(S) A ^(S) A ^(S) G ^(S) G ^(S) T ^(S) T^(S) A ^(S) T ^(S) C ^(S) A 118 J. Immun., 1995, 155, ^(S) A(2′MO)5490; 5′ UTR of alterna- tive mRNA) 13101 Exon 4 (Borriello et al.; C^(S) T ^(S) T ^(S) T ^(S) G ^(S) G ^(S) A ^(S) G ^(S) A ^(S) T ^(S) T^(S) A ^(S) T ^(S) T ^(S) C ^(S) G ^(S) A ^(S) G ^(S) T ^(S) T 119 5′UTR of alternative mRNA) (2′MO) 13102 Exon 5 (Borriello et al.; G ^(S) C^(S) A ^(S) A ^(S) G ^(S) T ^(S) G ^(S) T ^(S) A ^(S) A ^(S) A ^(S) G^(S) C ^(S) C ^(S) C ^(S) T ^(S) G ^(S) A ^(S) G ^(S) T 120 5′UTR of alternative mRNA) (2′MO)cDNA Clones:

A cDNA encoding the sequence for human B7-1 was isolated by using thereverse transcription/polymerase chain reaction (RT-PCR). Poly A+ RNAfrom Daudi cells (ATCC accession No. CCL 213) was reverse transcribedusing oligo-dT primer under standard conditions. Following a 30 minutereaction at 42° C. and heat inactivation, the reaction mixture (20 μL)was brought to 100 μL with water. A 10 μL aliquot from the RT reactionwas then amplified in a 50 μL PCR reaction using the 5′ primer,

the 5′ primer, 5′-GAT-CAG-GGT-ACC-CCA-AAG-AAA-AAG-TGA-TTT-GTC- ATT-GC-3′(sense, SEQ ID NO: 20), and the 3′ primer,5′-GAT-AGC-CTC-GAG-GAT-AAT-GAA-TTG-GCT-GAC-AAG- AC-3′(antisense, SEQ ID NO: 21)

The primers included unique restriction sites for subcloning of the PCRproduct into the vector pcDNA-3 (Invitrogen, San Diego, Calif.). The 5′primer was designed to have identity with bases 1 to 26 of the publishedhuman B7-1 sequence (Freeman et al., J. Immunol., 1989, 143, 2714;positions 13-38 of the primer) and includes a Kpn I restriction site(positions 7-12 of the primer) for use in cloning. The 3′ primer wasdesigned to be complementary to bases 1450 to 1471 of the publishedsequence for B7-1 (positions 14-35 of the primer) and includes a Xho Irestriction site (positions 7-12 of the primer). Following PCR, thereaction was extracted with phenol and precipitated using ethanol. Theproduct was digested with the appropriate restriction enzymes and thefull-length fragment purified by agarose gel and ligated into the vectorpcDNA-3 (Invitrogen, San Diego, Calif.) prepared by digesting with thesame enzymes. The resultant construct, pcB7-1, was confirmed byrestriction mapping and DNA sequence analysis using standard procedures.A mouse B7-1 clone, pcmB7-1, was isolated in a similar manner by RT-PCRof RNA isolated from a murine B-lymphocyte cell line, 70Z3.

A cDNA encoding the sequence for human B7-2, position 1 to 1391, wasalso isolated by RT-PCR. Poly A+ RNA from Daudi cells (ATCC accessionNo. CCL 213) was reverse transcribed using oligo-dT primer understandard conditions. Following a 30 minute reaction at 42° C. and heatinactivation, the reaction mixture (20 μL) was brought to 100 μL withwater. A 10 μL aliquot from the RT reaction was then amplified in a 50μL PCR reaction using

the 5′ primer, 5′-GAT-CAG-GGT-ACC-AGG-AGC-CTT-AGG-AGG-TAC-GG-3′(sense, SEQ ID NO: 1), and  the 3′ primer,5′-GAT-AGC-CTC-GAG-TTA-TTT-CCA-GGT-CAT-GAG-CCA-3′(antisense, SEQ ID NO: 2).

The 5′ primer was designed to have identity with bases 1-20 of thepublished B7-2 sequence (Azuma et al., Nature, 1993, 366, 76 and GenbankAccession No. L25259; positions 13-32 of the primer) and includes a KpnI site (positions 7-12 of the primer) for use in cloning. The 3′ primerwas designed to have complementarity to bases 1370-1391 of the publishedsequence for B7-2 (positions 13-33 of the primer) and includes an Xho Irestriction site (positions 7-12 of the primer). Following PCR, thereaction was extracted with phenol and precipitated using ethanol. Theproduct was digested with Xho I and Kpn I, and the full-length fragmentpurified by agarose gel and ligated into the vector pcDNA-3 (Invitrogen,San Diego, Calif.) prepared by digesting with the same enzymes. Theresultant construct, pcB7-2, was confirmed by restriction mapping andDNA sequence analysis using standard procedures.

A mouse B7-2 clone, pcmB7-2, was isolated in a similar manner by RT-PCRof RNA isolated from P388D1 cells using the 5′ primer,

the 5′ primer, 5′-GAT-CAG-GGT-ACC-AAG-AGT-GGC-TCC-TGT-AGG-CA (sense, SEQ ID NO: 99), and   the 3′ primer,5′-GAT-AGC-CTC-GAG-GTA-GAA-TTC-CAA-TCA-GCT-GA (antisense, SEQ ID NO: 100).

The 5′ primer has identity with bases 1-20, whereas the 3′ primer iscomplementary to bases 1096-1115, of the published murine B7-2 sequence(Chen et al., J. Immun., 1994, 152, 4929). Both primers incorporate therespective restriction enzyme sites found in the other 5′ and 3′ primersused to prepare cDNA clones. The RT-PCR product was restricted with XhoI and Kpn I and ligated into pcDNA-3 (Invitrogen, Carlsbad, Calif.).

Other cDNA clones, corresponding to mRNAs resulting from alternativesplicing events, are cloned in like fashion, using primers containingthe appropriate restriction sites and having identity with (5′ primers),or complementarity to (3′ primers), the selected B7 mRNA.

Example 2 Modulation of hB7-1 Expression by Oligonucleotides

The ability of oligonucleotides to inhibit B7-1 expression was evaluatedby measuring the cell surface expression of B7-1 in transfected COS-7cells by flow cytometry.

Methods:

A T-175 flask was seeded at 75% confluency with COS-7 cells (ATCCaccession No. CRL 1651). The plasmid pcB7-1 was introduced into cells bystandard calcium phosphate transfection. Following a 4 hourtransfection, the cells were trypsinized and seeded in 12-well dishes at80% confluency. The cells were allowed to adhere to the plastic for 1hour and were then washed with phosphate-buffered saline (PBS). OptiMEM™(GIBCO-BRL, Gaithersburg, Md.) medium was added along with 15 μg/mL ofLipofectin™ (GIBCO-BRL, Gaithersburg, Md.) and oligonucleotide at theindicated concentrations. After four additional hours, the cells werewashed with phosphate buffered saline (PBS) and incubated with fresholigonucleotide at the same concentration in DMEM (Dulbecco et al.,Virol., 1959, 8, 396; Smith et al., Virol., 1960, 12, 185) with 10%fetal calf sera (FCS).

In order to monitor the effects of oligonucleotides on cell surfaceexpression of B7-1, treated COS-7 cells were harvested by brieftrypsinization 24-48 hours after oligonucleotide treatment. The cellswere washed with PBS, then resuspended in 100 μL of staining buffer(PBS, 0.2% BSA, 0.1% azide) with 5 μL conjugated anti-B7-1-antibody(i.e., anti-hCD80-FITC, Ancell, Bayport, Minn.; FITC: fluoresceinisothiocyanate). The cells were stained for 30 minutes at 4° C., washedwith PBS, resuspended in 300 μL containing 0.5% paraformaldehyde. Cellswere harvested and the fluorescence profiles were determined using aflow cytometer.

Results:

The oligonucleotides shown in Table 1 were evaluated, in COS-7 cellstransiently expressing B7-1 cDNA, for their ability to inhibit B7-1expression. The results (FIG. 1) identified ISIS 13805, targeted to thetranslation initiation codon region, and ISIS 13812, targeted to the 3′untranslated region (UTR), as the most active oligonucleotides withgreater than 50% inhibition of B7-1 expression. These oligonucleotidesare thus highly preferred. ISIS 13799 (targeted to the 5′ untranslatedregion), ISIS 13802 (targeted to the 5′ untranslated region), ISIS 13806and 13807 (both targeted to the 5′ region of the ORF), and ISIS 13810(targeted to the central portion of the ORF) demonstrated 35% to 50%inhibition of B7-1 expression. These sequences are therefore alsopreferred. Oligonucleotide ISIS 13800, which showed essentially noinhibition of B7-1 expression in the flow cytometry assay, and ISIS Nos.13805 and 13812 were then evaluated for their ability to inhibit cellsurface expression of B7-1 at various concentrations of oligonucleotide.The results of these assays are shown in FIG. 2. ISIS 13812 was asuperior inhibitor of B7-1 expression with an IC₅₀ of approximately 150nM. ISIS 13800, targeted to the 5′ UTR, was essentially inactive.

Example 3 Modulation of hB7-2 Protein by Oligonucleotides

In an initial screen, the ability of hB7-2 oligonucleotides to inhibitB7-2 expression was evaluated by measuring the cell surface expressionof B7-2 in transfected COS-7 cells by flow cytometry. The methods usedwere similar to those given in Example 2, with the exceptions that (1)COS-7 cells were transfected with the plasmids pbcB7-2 or BBG-58, ahuman ICAM-1 (CD54) expression vector (R&D Systems, Minneapolis, Minn.)introduced into cells by standard calcium phosphate transfection, (2)the oligonucleotides used were those described in Table 2, and (3) aconjugated anti-B7-2 antibody (i.e., anti-hCD86-FITC or anti-CD86-PE,PharMingen, San Diego, Calif.; PE: phycoerythrin) was used during flowcytometry.

Results:

The results are shown in FIG. 3. At a concentration of 200 nM, ISIS9133, ISIS 9139 and ISIS 10373 exhibited inhibitory activity of 50% orbetter and are therefore highly preferred. These oligonucleotides aretargeted to the 3′ untranslated region (ISIS 9133), the translationinitiation codon region (ISIS 9139) and the 5′ untranslated region (ISIS10373). At the same concentration, ISIS 10715, ISIS 10716 and ISIS10721, which are scrambled controls for ISIS 9133, ISIS 9139 and ISIS10373, respectively, showed no inhibitory activity. Treatment with ISIS10367 and ISIS 10369 resulted in greater than 25% inhibition, and theseoligonucleotides are thus also preferred. These oligonucleotides aretargeted to the 5′ (ISIS 10367) and 3′(ISIS 10369) untranslated regions.

Example 4 Modulation of hB7-2 mRNA by Oligonucleotides

Methods:

For ribonuclease protection assays, cells were harvested 18 hours aftercompletion of oligonucleotide treatment using a Totally RNA™ kit(Ambion, Austin, Tex.). The probes for the assay were generated fromplasmids pcB7-2 (linearized by digestion with Bgl II) and pTRI-b-actin(Ambion Inc., Austin, Tex.). In vitro transcription of the linearizedplasmid from the SP6 promoter was performed in the presence of α-³²P-UTP(800 Ci/mmole) yielding an antisense RNA complementary to the 3′ end ofB7-2 (position 1044-1391). The probe was gel-purified after treatmentwith DNase I to remove DNA template. Ribonuclease protection assays werecarried out using an RPA II™ kit (Ambion) according to themanufacturer's directions. Total RNA (5 μg) was hybridized overnight, at42° C., with 10⁵ cpm of the B7-2 probe or a control beta-actin probe.The hybridization reaction was then treated, at 37° C. for 30 minutes,with 0.4 units of RNase A and 2 units of RNase T1. Protected RNA wasprecipitated, resuspended in 10 μL of gel loading buffer andelectrophoresed on a 6% acrylamide gel with 50% w/v urea at 20 W. Thegel was then exposed and the lanes quantitated using a PhosphorImager(Molecular Dynamics, Sunnyvale, Calif.) essentially according to themanufacturer's instructions.

Results:

The extent of oligonucleotide-mediated hB7-2 mRNA modulation generallyparalleled the effects seen for hB7-2 protein (Table 5). As with theprotein expression (flow cytometry) assays, the most activeoligonucleotides were ISIS 9133, ISIS 9139 and 10373. None of theoligonucleotides tested had an inhibitory effect on the expression ofb-actin mRNA in the same cells.

TABLE 5 Activities of Oligonucleotides Targeted to hB7-2 mRNA % Control% Control RNA ISIS NO. SEQ ID NO. Protein Expression 9133 3 70.2 46.09134 4 88.8 94.5 9135 5 98.2 83.4 9136 6 97.1 103.1 9137 7 80.5 78.19138 8 86.4 65.9 9139 9 47.9 32.6 10367 10 71.3 52.5 10368 11 81.0 84.510369 12 71.3 81.5 10370 13 84.3 83.2 10371 14 97.3 92.9 10372 15 101.782.5 10373 16 43.5 32.7

Example 5 Additional hB7-1 and hB7-2 Oligonucleotides

Oligonucleotides having structures and/or sequences that were modifiedrelative to the oligonucleotides identified during the initial screeningwere prepared. These oligonucleotides were evaluated for their abilityto modulate human B7-2 expression using the methods described in theprevious examples. ISIS 10996, an oligonucleotide having a 15 nucleotidesequence derived from the 20 nucleotide sequence of ISIS 10373, was alsoprepared and evaluated. ISIS 10996 comprises 15 nucleotides,5′-GCG-AGC-TCC-CCG-TAC (SEQ ID NO: 90) contained within the sequence ofISIS 10373. Both ISIS 10373 and 10996 overlap a potential stem-loopstructure located within the B7-2 message comprising bases 1-67 of thesequence of hB7-2 presented by Azuma et al. (Nature, 1993, 366, 76).While not intending to be bound be any particular theory regarding theirmode(s) of action, ISIS 10373 and ISIS 10996 have the potential to bindas loop 1 pseudo-half-knots at a secondary structure within the targetRNA. U.S. Pat. No. 5,5152,438, the contents of which are herebyincorporated by reference, describes methods for modulating geneexpression by the formation of pseudo-half-knots. Regardless of theirmode(s) of action, despite having a shorter length than ISIS 10373, the15-mer ISIS 10886 is as (or more) active in the B7-2 protein expressionassay than the 20-mer from which it is derived (FIG. 4; ISIS 10721 is ascrambled control for ISIS 10373). A related 16-mer, ISIS 10998, wasalso active in the B7-2 protein expression assay. However, astructurally related 14-mer (ISIS 10995), 13-mer (ISIS 10994), 12-mer(ISIS 10993), 11-mer (ISIS 10992) and 10-mer (ISIS 10991) exhibitedlittle or no activity in this assay. ISIS 10996 was further derivatizedin the following ways.

ISIS 10996 derivatives having 2′ methoxyethoxy substitutions wereprepared, including a fully substituted derivative (ISIS 11539),“gapmers” (ISIS 11541 and 11543) and “wingmers” (ISIS 11545 and 11547).As explained in Example 5, the 2′ methoxyethoxy substitution preventsthe action of some nucleases (e.g., RNase H) but enhances the affinityof the modified oligonucleotide for its target RNA molecule. Theseoligonucleotides are tested for their ability to modulate hB7-2 messageor function according to the methods of Examples 3, 4, 7 and 8.

ISIS 10996 derivatives were prepared in order to be evaluated for theirability to recruit RNase L to a target RNA molecule, e.g., hB7-2message. RNase L binds to, and is activated by, (2′-5′)(A)_(n), which isin turn produced from ATP by (2′-5′)(A)_(n) synthetase upon activationby, e.g., interferon. RNase L has been implicated in antiviralmechanisms and in the regulation of cell growth as well (Sawai, ChemicaScripta, 1986, 21, 169; Charachon et al., Biochemistry, 1990, 29, 2550).The combination of anti-B7 oligonucleotides conjugated to(2′-5′)(A)_(n), is expected to result in the activation of RNase L andits targeting to the B7 message complementary to the oligonucleotidesequence. The following oligonucleotides have identical sequences (i.e.,that of ISIS 10996) and identical (2′-5′)(A)₄ “caps” on their 5′termini: ISIS 12492, 12495, 12496 and 13107. The adenosyl residues have3′ hydroxyl groups and are linked to each other by phosphorothioatelinkages. The (3′-5′) portion of the oligonucleotide, which has asequence complementary to a portion of the human B7-2 RNA, is conjugatedto the (2′-5′)(A)₄ “cap” via a phosphorothioate linkage from the 5′residue of the (3′-5′) portion of the oligonucleotide to an n-aminohexyllinker which is bonded to the “cap” via another phosphorothioatelinkage. In order to test a variety of chemically diverseoligonucleotides of this type for their ability to recruit RNase L to aspecific message, different chemical modifications were made to this setof four oligonucleotides as follows. ISIS 12496 consists of unmodifiedoligonucleotides in the (3′-5′) portion of the oligonucleotide. In ISIS13107, phosphorothioate linkages replace the phosphate linkages found innaturally occurring nucleic acids. Phosphorothioate linkages are alsoemployed in ISIS 12492 and 12495, which additionally have2′-methoxyethoxy substitutions. These oligonucleotides are tested fortheir ability to modulate hB7-2 message or function according to themethods of Examples 3, 4, 7 and 8.

Derivatives of ISIS 10996 having modifications at the 2′ position wereprepared and evaluated. The modified oligonucleotides included ISIS11539 (fully 2′-O-methyl), ISIS 11541 (having 2′-O-methyl wings and acentral 7-base gap), ISIS 11543 (2′-O-methyl wings with a 9-base gap),ISIS 11545 (having a 5′2′-O-methyl wing) and ISIS 11547 (having a3′2′-O-methyl wing). The results of assays of 2′-O-methyloligonucleotides were as follows. ISIS 11539, the fully 2′O-methylversion of ISIS 10996, was not active at all in the protein expressionassay. The gapped and winged oligonucleotides (ISIS 11541, 11543, 11545and 11547) each showed some activity at 200 nM (i.e., from 60 to 70%expression relative to untreated cells), but less than that demonstratedby the parent compound, ISIS 10996 (i.e., about 50% expression). Similarresults were seen in RNA expression assays.

ISIS 10782, a derivative of ISIS 10373 to which cholesterol has beenconjugated via a 5′ n-aminohexyl linker, was prepared. Lipophilicmoieties such as cholesterol have been reported to enhance the uptake bycells of oligonucleotides in some instances, although the extent towhich uptake is enhanced, if any, remains unpredictable. ISIS 10782, andother oligonucleotides comprising lipophilic moieties, are tested fortheir ability to modulate B7-2 message or function according to themethods of Examples 3, 4, 7 and 8.

A series of 2′-methoxyethoxy (herein, “2′ME”) and 2′-fluoride (herein,“2′F”) “gapmer” derivatives of the hB7-1 oligonucleotides ISIS 12361(ISIS Nos. 12348 and 12473, respectively), ISIS 12362 (ISIS Nos. 12349and 12474), ISIS 12363 (ISIS Nos. 12350 and 12475), ISIS 12364 (ISISNos. 12351 and 12476), ISIS 12365 (ISIS Nos. 12352 and 12477), ISIS12366 (ISIS Nos. 12353 and 12478), ISIS 12367 (ISIS Nos. 12354 and12479), ISIS 12368 (ISIS Nos. 12355 and 12480), ISIS 12369 (ISIS Nos.12356 and 12481) and ISIS 12370 (ISIS Nos. 12357 and 12482) wereprepared. The central, non-2′-modified portions (“gaps”) of thesederivatives support RNase H activity when the oligonucleotide is boundto its target RNA, even though the 2′-modified portions do not. However,the 2′-modified “wings” of these oligonucleotides enhance their affinityto their target RNA molecules (Cook, Chapter 9 In: Antisense Researchand Applications, Crooke et al., eds., CRC Press, Boca Raton, 1993, pp.171-172).

Another 2′ modification is the introduction of a methoxy (MO) group atthis position. Like 2′ME- and 2′F-modified oligonucleotides, thismodification prevents the action of RNase H on duplexes formed from sucholigonucleotides and their target RNA molecules, but enhances theaffinity of an oligonucleotide for its target RNA molecule. ISIS 12914and 12915 comprise sequences complementary to the 5′ untranslated regionof alternative hB7-1 mRNA molecules, which arise from alternativesplicing events of the primary hB7-1 transcript. These oligonucleotidesinclude 2′ methoxy modifications, and the enhanced target affinityresulting therefrom may allow for greater activity against alternativelyspliced B7-1 mRNA molecules which may be present in low abundance insome tissues (Inobe et al., J. Immun., 1996, 157, 582). Similarly, ISIS13498 and 13499, which comprise antisense sequences to other alternativehB7-1 mRNAs, include 2′ methoxyethoxy modifications in order to enhancetheir affinity for their target molecules, and 2′ methoxyethoxy or2′methoxy substitutions are incorporated into the hB7-2 oligonucleotidesISIS 12912, 12913, 13496 and 13497. These oligonucleotides are testedfor their ability to modulate hB7-1 essentially according to the methodsof Example 2 or hB7-2 according to the methods of Examples 3, 4, 7 and8, with the exception that, when necessary, the target cells aretransfected with a cDNA clone corresponding to the appropriatealternatively spliced B7 transcript.

Example 6 Specificity of Antisense Modulation

Several oligonucleotides of the invention were evaluated in a cellsurface expression flow cytometry assay to determine the specificity ofthe oligonucleotides for B7-1 as contrasted with activity against B7-2.The oligonucleotides tested in this assay included ISIS 13812, aninhibitor of B7-1 expression (FIG. 1; Example 2) and ISIS 10373, aninhibitor of B7-2 expression (FIG. 3; Example 3). The results of thisassay are shown in FIG. 5. ISIS 13812 inhibits B7-1 expression withlittle or no effect on B7-2 expression. As is also seen in FIG. 5, ISIS10373 inhibits B7-2 expression with little or no effect on B7-1expression. ISIS 13872 (SEQ ID NO: 37, AGT-CCT-ACT-ACC-AGC-CGC-CT), ascrambled control of ISIS 13812, and ISIS 13809 (SEQ ID NO: 51) wereincluded in these assays and demonstrated essentially no activityagainst either B7-1 or B7-2.

Example 7 Modulation of hB7-2 Expression by Oligonucleotides in AntigenPresenting Cells

The ability of ISIS 10373 to inhibit expression from the native B7-2gene in antigen presenting cells (APCs) was evaluated as follows.

Methods:

Monocytes were cultured and treated with oligonucleotides as follows.For dendritic cells, EDTA-treated blood was layered onto Polymorphprep™(1.113 g/mL; Nycomed, Oslo, Norway) and sedimented at 500×g for 30minutes at 20° C. Mononuclear cells were harvested from the interface.Cells were washed with PBS, with serum-free RPMI media (Moore et al.,N.Y. J. Med., 1968, 68, 2054) and then with RPMI containing 5% fetalbovine serum (FBS). Monocytes were selected by adherence to plastic cellculture cell culture dishes for 1 h at 37° C. After adherence, cellswere treated with oligonucleotides in serum-free RPMI containingLipofectin™ (8 μg/mL). After 4 hours, the cells were washed. Then RPMIcontaining 5% FBS and oligonucleotide was added to cells along withinterleukin-4 (IL-4; R&D Systems, Minneapolis, Minn.) (66 ng/mL) andgranulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Systems,Minneapolis, Minn.) (66 ng/mL) to stimulate differentiation (Romani etal., J. Exp. Med., 1994, 180, 83, 1994). Cells were incubated for 48hours, after which cell surface expression of various molecules wasmeasured by flow cytometry.

Mononuclear cells isolated from fresh blood were treated witholigonucleotide in the presence of cationic lipid to promote cellularuptake. As a control oligonucleotide, ISIS 2302 (an inhibitor of ICAM-1expression; SEQ ID NO: 17) was also administered to the cells.Expression of B7-2 protein was measured by flow cytometry according tothe methods of Example 2. Monoclonal antibodies not described in theprevious Examples included anti-hCD3 (Ancell, Bayport, Minn.) andanti-HLA-DR (Becton Dickinson, San Jose, Calif.).

Results:

As shown in FIG. 6, ISIS 10373 has a significant inhibitory effect onB7-2 expression with an IC₅₀ of approximately 250 nM. ISIS 10373 hadonly a slight effect on ICAM-1 expression even at a dose of 1 μM. ISIS2302 (SEQ ID NO: 17), a control oligonucleotide which has been shown toinhibit ICAM-1 expression, had no effect on B7-2 expression, butsignificantly decreased ICAM-1 levels with an IC₅₀ of approximately 250nM. Under similar conditions, ISIS 10373 did not affect the cell surfaceexpression of B7-1, HLA-DR or CD3 as measured by flow cytometry.

Example 8 Modulation of T Cell Proliferation by Oligonucleotides

The ability of ISIS 2302 and ISIS 10373 to inhibit T cell proliferationwas evaluated as follows. Monocytes treated with oligonucleotide andcytokines (as in Example 6) were used as antigen presenting cells in a Tcell proliferation assay. The differentiated monocytes were combinedwith CD4+ T cells from a separate donor. After 48 hours, proliferationwas measured by [³H]thymidine incorporation.

Methods:

For T cell proliferation assays, cells were isolated from EDTA-treatedwhole blood as described above, except that a faster migrating bandcontaining the lymphocytes was harvested from just below the interface.Cells were washed as described in Example 6 after which erythrocyteswere removed by NH₄Cl lysis. T cells were purified using a T cellenrichment column (R&D Systems, Minneapolis, Minn.) essentiallyaccording to the manufacturer's directions. CD4+ T cells were furtherenriched from the entire T cell population by depletion of CD8+ cellswith anti-CD8-conjugated magnetic beads (AMAC, Inc., Westbrook, Me.)according to the manufacturer's directions. T cells were determined tobe >80% CD4+ by flow cytometry using Cy-chrome-conjugated anti-CD4 mAb(PharMingen, San Diego, Calif.).

Antigen presenting cells (APCs) were isolated as described in Example 6and treated with mitomycin C (25 μg/mL) for 1 hour then washed 3 timeswith PBS. APCs (10⁵ cells) were then combined with 4×10⁴ CD4+ T cells in350 μL of culture media. Where indicated, purified CD3 mAb was alsoadded at a concentration of 1 μg/mL. During the last 6 hours of the 48hour incubation period, proliferation was measured by determining uptakeof 1.5 μCi of [³H]-thymidine per well. The cells were harvested ontofilters and the radioactivity measured by scintillation counting.

Results:

As shown in FIG. 7, mononuclear cells which were not cytokine-treatedslightly induced T cell proliferation, presumably due to low levels ofcostimulatory molecules expressed on the cells. However, when the cellswere treated with cytokines and induced to differentiate todendritic-like cells, expression of both ICAM-1 and B7-2 was stronglyupregulated. This resulted in a strong T cell proliferative responsewhich could be blocked with either anti-ICAM-1 (ISIS 2302) or anti-B7-2(ISIS 10373) oligonucleotides prior to induction of the mononuclearcells. The control oligonucleotide (ISIS 10721) had an insignificanteffect on T cell proliferation. A combination treatment with both theanti-ICAM-1 (ISIS 2302) and anti-B7-2 (ISIS 10373) oligonucleotidesresulted in a further decrease in T cell response.

Example 9 Modulation of Murine B7 Genes by Oligonucleotides

Oligonucleotides (see Table 4) capable of inhibiting expression ofmurine B7-2 transiently expressed in COS-7 cells were identified in thefollowing manner. A series of phosphorothioate oligonucleotidescomplementary to murine B7-2 (mB7-2) cDNA were screened for theirability to reduce mB7-2 levels (measured by flow cytometry as in Example2, except that a conjugated anti-mB7-2 antibody (i.e., anti-mCD86-PE,PharMingen, San Diego, Calif.) in COS-7 cells transfected with an mB7-2cDNA clone. Anti-mB7-2 antibody may also be obtained from the hybridomadeposited at the ATCC under accession No. HB-253. Oligonucleotides (seeTable 2) capable of modulating murine B7-1 expression are isolated inlike fashion, except that a conjugated anti-mB7-1 antibody is used inconjunction with COS-7 cells transfected with an mB7-1 cDNA clone.

For murine B7-2, the most active oligonucleotide identified was ISIS11696 (GGA-TTG-CCA-AGC-CCA-TGG-TG, SEQ ID NO: 18), which iscomplementary to position 96-115 of the cDNA, a site which includes thetranslation initiation (AUG) codon. FIG. 8 shows a dose-response curvefor ISIS 11696 and a scrambled control, ISIS 11866(CTA-AGT-AGT-GCT-AGC-CGG-GA, SEQ ID NO: 19). ISIS 11696 inhibited cellsurface expression of B7-2 in COS-7 cells with an IC₅₀ in the range of200-300 nM, while ISIS 11866 exhibited less than 20% inhibition at thehighest concentration tested (1000 nM).

In order to further evaluate the murine B7-2 antisense oligonucleotides,the IC-21 cell line was used. IC-21 monocyte/macrophage cell lineexpresses both B7-1 and murine B7-2 (mB7-2) constitutively. A 2-foldinduction of expression can be achieved by incubating the cells in thepresence of lipopolysaccharide (LPS; GIBCO-BRL, Gaithersburg, Md.)(Hathcock et al., Science, 1993, 262, 905).

IC-21 cells (ATCC; accession No. TIB 186) were seeded at 80% confluencyin 12-well plates in DMEM media with 10% FCS. The cells were allowed toadhere to the plate overnight. The following day, the medium was removedand the cells were washed with PBS. Then 500 μL of OptiMEM™ (GIBCO-BRL,Gaithersburg, Md.) supplemented with 15 μg/mL of Lipofectin™ (GIBCO-BRL,Gaithersburg, Md.) was added to each well. Oligonucleotides were thenadded directly to the medium at the indicated concentrations. Afterincubation for 4 hours, the cells were washed with PBS and incubatedovernight in culture medium supplemented with 15 μg/mL of LPS. Thefollowing day, cells were harvested by scraping, then analyzed for cellsurface expression by flow cytometry.

ISIS 11696 and ISIS 11866 were administered to IC-21 cells in thepresence of Lipofectin™ (GIBCO-BRL, Gaithersburg, Md.). The results areshown in FIG. 9. At a concentration of 10 μM, ISIS 11696 inhibited mB7-2expression completely (and decreased mB7-2 levels below the constitutivelevel of expression), while the scrambled control oligonucleotide, ISIS11866, produced only a 40% reduction in the level of induced expression.At a concentration of 3 μM, levels of induced expression were greatlyreduced by ISIS 11696, while ISIS 11866 had little effect.

Modified oligonucleotides, comprising 2′ substitutions (e.g., 2′methoxy, 2′ methoxyethoxy) and targeted to alternative transcripts ofmurine B7-1 (ISIS 12914, 12915, 13498, 13499) or murine B7-2 (ISIS13100, 13100 and 13102) were prepared. These oligonucleotides are testedfor their ability to modulate murine B7 essentially according to theabove methods using IC-21 cells or COS-7 transfected with a cDNA clonecorresponding to the appropriate alternatively spliced B7 transcript.

Example 10 Modulation of Allograft Rejection by Oligonucleotides

A murine model for evaluating compounds for their ability to inhibitheart allograft rejection has been previously described (Stepkowski etal., J. Immunol., 1994, 153, 5336). This model was used to evaluate theimmunosuppressive capacity of antisense oligonucleotides to B7 proteinsalone or in combination with antisense oligonucleotides to intercellularadhesion molecule-1 (ICAM-1).

Methods:

Heart allograft rejection studies and oligonucleotide treatments ofBALB/c mice were performed essentially as previously described(Stepkowski et al., J. Immunol., 1994, 153, 5336). Antisenseoligonucleotides used included ISIS 11696, ISIS 3082 (targeted toICAM-1) and ISIS 1082 (a control oligonucleotide targeted to the herpesvirus UL-13 gene sequence). Dosages used were 1, 2, 2.5, 5 or 10 mg/kgof individual oligonucleotide (as indicated below); when combinations ofoligonucleotides were administered, each oligonucleotide was given at adosage of 1, 5 or 10 mg/kg (total oligonucleotide dosages of 2, 10 and20 mg/kg, respectively). The survival times of the transplanted heartsand their hosts were monitored and recorded.

Results:

The mean survival time for untreated mice was 8.2±0.8 days (7, 8, 8, 8,9, 9 days). Treatment of the mice for 7 days with ISIS 1082 (SEQ ID NO:125, unrelated control oligonucleotide) slightly reduced the meansurvival times to 7.1±0.7 days (5 mg/kg/day; 6, 7, 7, 7, 8, 8) or7.0±0.8 days (10 mg/kg/day; 6, 7, 7, 8). Treatment of the mice for sevendays with the murine B7-2 oligonucleotide ISIS 11696 (SEQ ID NO: 108)increased the mean survival time to 9.3 days at two doses (2 mg/kg/day,9.3±0.6 days, 9, 9, 10; 10 mg/kg/day, 9.3±1.3 days, 8, 9, 9, 11).Treatment of mice for seven days with an ICAM-1 oligonucleotide, ISIS3082, also increased the mean survival of the mice over several doses.Specifically, at 1 mg/kg/day, the mean survival time (MSD) was 11.0±0.0(11, 11, 11); at 2.5 mg/kg/day, the MSD was 12.0±2.7 (10, 12, 13, 16);at 5 mg/kg/day, the MSD was 14.1±2.7 (10, 12, 12, 13, 16, 16, 17, 17);and, at 10 mg/kg/day, the MSD was 15.3±5.8 (12, 12, 13, 24). Somesynergistic effect was seen when the mice were treated for seven dayswith 1 mg/kg/day each of ISIS 3082 and 11696: the MSD was 13.8±1.0 (13,13, 14, 15).

Example 11 Detection of Nucleic Acids Encoding B7 Proteins

Oligonucleotides are radiolabeled after synthesis by ³²P-labeling at the5′ end with polynucleotide kinase. Sambrook et al., “Molecular Cloning.A Laboratory Manual,” Cold Spring Harbor Laboratory Press, 1989, Volume2, pg. 11.31. Radiolabeled oligonucleotide capable of hybridizing to anucleic acid encoding a B7 protein is contacted with a tissue or cellsample suspected of B7 protein expression under conditions in whichspecific hybridization can occur, and the sample is washed to removeunbound oligonucleotide. A similar control is maintained wherein theradiolabeled oligonucleotide is contacted with a normal tissue or cellsample under conditions that allow specific hybridization, and thesample is washed to remove unbound oligonucleotide. Radioactivityremaining in the samples indicates bound oligonucleotide and isquantitated using a scintillation counter or other routine means. Agreater amount of radioactivity remaining in the samples, as compared tocontrol tissues or cells, indicates increased expression of a B7 gene,whereas a lesser amount of radioactivity in the samples relative to thecontrols indicates decreased expression of a B7 gene.

Radiolabeled oligonucleotides of the invention are also useful inautoradiography. A section of tissues suspected of expressing a B7 geneis treated with radiolabeled oligonucleotide and washed as describedabove, then exposed to photographic emulsion according to standardautoradiography procedures. A control of a normal tissue section is alsomaintained. The emulsion, when developed, yields an image of silvergrains over the regions expressing a B7 gene, which is quantitated. Theextent of B7 expression is determined by comparison of the silver grainsobserved with control and test samples.

Analogous assays for fluorescent detection of expression of a B7 geneuse oligonucleotides of the invention which are labeled with fluoresceinor other fluorescent tags. Labeled oligonucleotides are synthesized onan automated DNA synthesizer (Applied Biosystems, Foster City, Calif.)using standard phosphoramidite chemistry. b-Cyanoethyldiisopropylphosphoramidites are purchased from Applied Biosystems (Foster City,Calif.). Fluorescein-labeled amidites are purchased from Glen Research(Sterling, Va.). Incubation of oligonucleotide and biological sample iscarried out as described above for radiolabeled oligonucleotides exceptthat, instead of a scintillation counter, a fluorescence microscope isused to detect the fluorescence. A greater amount of fluorescence in thesamples, as compared to control tissues or cells, indicates increasedexpression of a B7 gene, whereas a lesser amount of fluorescence in thesamples relative to the controls indicates decreased expression of a B7gene.

Example 12 Chimeric (Deoxy Gapped) Human B7-1 Antisense Oligonucleotides

Additional oligonucleotides targeting human B7-1 were synthesized.Oligonucleotides were synthesized as uniformly phosphorothioate chimericoligonucleotides having regions of five 2′-O-methoxyethyl (2′-MOE)nucleotides at the wings and a central region of ten deoxynucleotides.Oligonucleotide sequences are shown in Table 6.

Oligonucleotides were screened as described in Example 4. Results areshown in Table 7.

Oligonucleotides 22315 (SEQ ID NO: 128), 22316 (SEQ ID NO: 26), 22317(SEQ ID NO: 129), 22320 (SEQ ID NO: 132), 22324 (SEQ ID NO: 135), 22325(SEQ ID NO: 136), 22334 (SEQ ID NO: 145), 22335 (SEQ ID NO: 146), 22337(SEQ ID NO: 148), and 22338 (SEQ ID NO: 36) resulted in 50% or greaterinhibition of B7-1 mRNA in this assay.

TABLE 6 Nucleotide Sequences of Human B7-1 Chimeric(deoxy gapped) Oligodeoxynucleotides SEQ TARGET GENE GENE ISISNUCLEOTIDE SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′->3′) NO: COORDINATES²REGION 22313 AGACTCCACTTCTGAGATGT 126 0048-0067 5′-UTR 22314TGAAGAAAAATTCCACTTTT 127 0094-0113 5′-UTR 22315 TTTAGTTTCACAGCTTGCTG 1280112-0129 5′-UTR 22316 GCTCACGTAGAAGACCCTCC 26 0193-0212 5′-UTR 22317TCCCAGGTGCAAAACAGGCA 129 0233-0252 5′-UTR 22318 GTGAAAGCCAACAATTTGGA 1300274-0293 5′-UTR 22319 CATGGCTTCAGATGCTTAGG 131 0301-0320 AUG 22320TTGAGGTATGGACACTTGGA 132 0351-0370 coding 22321 GACCAGCCAGCACCAAGAGC 310380-0399 coding 22322 GCGTTGCCACTTCTTTCACT 133 0440-0459 coding 22323TTTTGCCAGTAGATGCGAGT 134 0501-0520 coding 22324 GGCCATATATTCATGTCCCC 1350552-0571 coding 22325 GCCAGGATCACAATGGAGAG 136 0612-0631 coding 22326GTATGTGCCCTCGTCAGATG 137 0640-0659 coding 22327 TTCAGCCAGGTGTTCCCGCT 1380697-0716 coding 22328 GGAAGTCAGCTTTGACTGAT 139 0725-0744 coding 22329CCTCCAGAGGTTGAGCAAAT 140 0798-0817 coding 22330 CCAACCAGGAGAGGTGAGGC 1410827-0846 coding 22331 GAAGCTGTGGTTGGTTGTCA 142 0940-0959 coding 22332TTGAAGGTCTGATTCACTCT 143 0987-1006 coding 22333 AAGGTAATGGCCCAGGATGG 1441050-1069 coding 22334 AAGCAGTAGGTCAGGCAGCA 145 1098-1117 coding 22335CCTTGCTTCTGCGGACACTG 146 1185-1204 3′-UTR 22336 AGCCCCTTGCTTCTGCGGAC 1471189-1208 3′-UTR 22337 TGACGGAGGCTACCTTCAGA 148 1216-1235 3′-UTR 22338GCCTCATGATCCCCACGATC 36 1254-1273 3′-UTR 22339 GTAAAACAGCTTAAATTTGT 1491286-1305 3′-UTR 22340 AGAAGAGGTTACATTAAGCA 150 1398-1417 3′-UTR 22341AGATAATGAATTGGCTGACA 151 1454-1473 3′-UTR 24733 GCGTCATCATCCGCACCATC 152control 24734 CGTTGCTTGTGCCGACAGTG 153 control 24735GCTCACGAAGAACACCTTCC 154 control ¹Emboldened residues are2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethylcytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; alllinkages are phosphorothioate linkages. ²Co-ordinates from GenbankAccession No. M27533, locus name “HUMIGB7”.

TABLE 7 Inhibition of Human B7-1 mRNA Expression byChimeric (deoxy gapped) Phosphorothioate Oligodeoxynucleotides SEQ GENEISIS ID TARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION basal— — 100% — 13805 30 AUG 46% 54% 13812 36 3′-UTR 22% 78% 22313 126 5′-UTR75% 25% 22314 127 5′-UTR 69% 31% 22315 128 5′-UTR 49% 51% 22316 265′-UTR 42% 58% 22317 129 5′-UTR 43% 57% 22318 130 5′-UTR 63% 37% 22319131 AUG 68% 32% 22320 132 coding 45% 55% 22321 31 coding 57% 43% 22324135 coding 46% 54% 22325 136 coding 46% 54% 22326 137 coding 62% 38%22328 139 coding 64% 36% 22329 140 coding 59% 41% 22330 141 coding 54%46% 22331 142 coding 62% 38% 22332 143 coding 67% 33% 22333 144 coding73% 27% 22334 145 coding 43% 57% 22335 146 3′-UTR 43% 57% 22336 1473′-UTR 55% 45% 22337 148 3′-UTR 42% 58% 22338 36 3′-UTR 40% 60% 22339149 3′-UTR 69% 31% 22340 150 3′-UTR 71% 29% 22341 151 3′-UTR 59% 41%

Dose response experiments were performed on several of the more activeoligonucleotides. The oligonucleotides were screened as described inExample 4 except that the concentration of oligonucleotide was varied asshown in Table 8. Mismatch control oligonucleotides were included.Results are shown in Table 8.

All antisense oligonucleotides tested showed a dose response effect withinhibition of mRNA approximately 60% or greater.

TABLE 8 Dose Response of COS-7 Cells to B7-1 Chimeric(deoxy gapped) Antisense Oligonucleotides SEQ ID Gene % mRNA % mRNAISIS # NO: Target Dose Expression Inhibition basal — — — 100% — 22316 265′-UTR  10 nM 99% 1% ″ ″ ″  30 nM 73% 27% ″ ″ ″ 100 nM 58% 42% ″ ″ ″300 nM 33% 67% 24735 154 control  10 nM 100% — ″ ″ ″  30 nM 95% 5% ″ ″ ″100 nM 81% 19% ″ ″ ″ 300 nM 75% 25% 22335 146 3′-UTR  10 nM 81% 19% ″ ″″  30 nM 63% 37% ″ ″ ″ 100 nM 43% 57% ″ ″ ″ 300 nM 35% 65% 24734 153control  10 nM 94% 6% ″ ″ ″  30 nM 96% 4% ″ ″ ″ 100 nM 94% 6% ″ ″ ″300 nM 84% 16% 22338 36 3′-UTR  10 nM 68% 32% ″ ″ ″  30 nM 60% 40% ″ ″ ″100 nM 53% 47% ″ ″ ″ 300 nM 41% 59% 24733 152 control  10 nM 90% 10% ″ ″″  30 nM 91% 9% ″ ″ ″ 100 nM 90% 10% ″ ″ ″ 300 nM 80% 20%

Example 13 Chimeric (Deoxy Gapped) Mouse B7-1 Antisense Oligonucleotides

Additional oligonucleotides targeting mouse B7-1 were synthesized.Oligonucleotides were synthesized as uniformly phosphorothioate chimericoligonucleotides having regions of five 2′-O-methoxyethyl (2′-MOE)nucleotides at the wings and a central region of ten deoxynucleotides.Oligonucleotide sequences are shown in Table 9.

Oligonucleotides were screened as described in Example 4. Results areshown in Table 10. Oligonucleotides 18105 (SEQ ID NO: 156), 18106 (SEQID NO: 157), 18109 (SEQ ID NO: 160), 18110 (SEQ ID NO: 161), 18111 (SEQID NO: 162), 18112 (SEQ ID NO: 163), 18113 (SEQ ID NO: 164), 18114 (SEQID NO: 165), 18115 (SEQ ID NO: 166), 18117 (SEQ ID NO: 168), 18118 (SEQID NO: 169), 18119 (SEQ ID NO: 170), 18120 (SEQ ID NO: 171), 18122 (SEQID NO: 173), and 18123 (SEQ ID NO: 174) resulted in greater thanapproximately 50% inhibition of B7-1 mRNA in this assay.

TABLE 9 Nucleotide Sequences of Mouse B7-1 Chimeric(deoxy gapped) Oligodeoxynucleotides SEQ TARGET GENE GENE ISISNUCLEOTIDE SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′->3′) NO: CO-ORDINATES²REGION 18104 AGAGAAACTAGTAAGAGTCT 155 0018-0037 5′-UTR 18105TGGCATCCACCCGGCAGATG 156 0110-0129 5′-UTR 18106 TCGAGAAACAGAGATGTAGA 1570144-0163 5′-UTR 18107 TGGAGCTTAGGCACCTCCTA 158 0176-0195 5′-UTR 18108TGGGGAAAGCCAGGAATCTA 159 0203-0222 5′-UTR 18109 CAGCACAAAGAGAAGAATGA 1600310-0329 coding 18110 ATGAGGAGAGTTGTAACGGC 161 0409-0428 coding 18111AAGTCCGGTTCTTATACTCG 162 0515-0534 coding 18112 GCAGGTAATCCTTTTAGTGT 1630724-0743 coding 18113 GTGAAGTCCTCTGACACGTG 164 0927-0946 coding 18114CGAATCCTGCCCCAAAGAGC 165 0995-1014 coding 18115 ACTGCGCCGAATCCTGCCCC 1661002-1021 coding 18116 TTGATGATGACAACGATGAC 167 1035-1054 coding 18117CTGTTGTTTGTTTCTCTGCT 168 1098-1117 coding 18118 TGTTCAGCTAATGCTTCTTC 1691134-1153 coding 18119 GTTAACTCTATCTTGTGTCA 170 1263-1282 3′-UTR 18120TCCACTTCAGTCATCAAGCA 171 1355-1374 3′-UTR 18121 TGCTCAATACTCTCTTTTTA 1721680-1699 3′-UTR 18122 AGGCCCAGCAAACTTGCCCG 173 1330-1349 3′-UTR 18123AACGGCAAGGCAGCAATACC 174 0395-0414 coding ¹Emboldened residues are2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethylcytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; alllinkages are phosphorothioate linkages. ²Co-ordinates from GenbankAccession No. X60958, locus name “MMB7BLAA”.

TABLE 10 Inhibition of Mouse B7-1 mRNA Expression byChimeric (deoxy gapped) Phosphorothioate Oligodeoxynucleotides GENE ISISSEQ ID TARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION basal —— 100.0% — 18104 155 5′-UTR 60.0% 40.0% 18105 156 5′-UTR 32.0% 68.0%18106 157 5′-UTR 51.0% 49.0% 18107 158 5′-UTR 58.0% 42.0% 18108 1595′-UTR 82.0% 18.0% 18109 160 coding 45.5% 54.5% 18110 161 coding 21.0%79.0% 18111 162 coding 38.0% 62.0% 18112 163 coding 42.0% 58.0% 18113164 coding 24.6% 75.4% 18114 165 coding 25.6% 74.4% 18115 166 coding33.5% 66.5% 18116 167 coding 65.6% 34.4% 18117 168 coding 46.7% 53.3%18118 169 coding 31.7% 68.3% 18119 170 3′-UTR 24.0% 76.0% 18120 1713′-UTR 26.7% 73.3% 18121 172 3′-UTR 114.0% — 18122 173 3′-UTR 42.0%58.0% 18123 174 coding 42.0% 58.0%

Example 14 Chimeric (Deoxy Gapped) Human B7-2 Antisense Oligonucleotides

Additional oligonucleotides targeting human B7-2 were synthesized.Oligonucleotides were synthesized as uniformly phosphorothioate chimericoligonucleotides having regions of five 2′-O-methoxyethyl (2′-MOE)nucleotides at the wings and a central region of ten deoxynucleotides.Oligonucleotide sequences are shown in Table 11.

Oligonucleotides were screened as described in Example 4. Results areshown in Table 12. Oligonucleotides 22284 (SEQ ID NO: 16), 22286 (SEQ IDNO: 176), 22287 (SEQ ID NO: 177), 22288 (SEQ ID NO: 178), 22289 (SEQ IDNO: 179), 22290 (SEQ ID NO: 180), 22291 (SEQ ID NO: 181), 22292 (SEQ IDNO: 182), 22293 (SEQ ID NO: 183), 22294 (SEQ ID NO: 184), 22296 (SEQ IDNO: 186), 22299 (SEQ ID NO: 189), 22300 (SEQ ID NO: 190), 22301 (SEQ IDNO: 191), 22302 (SEQ ID NO: 192), 22303 (SEQ ID NO: 193), 22304 (SEQ IDNO: 194), 22306 (SEQ ID NO: 196), 22307 (SEQ ID NO: 197), 22308 (SEQ IDNO: 198), 22309 (SEQ ID NO: 199), 22310 (SEQ ID NO: 200), and 22311 (SEQID NO: 201) resulted in greater than 50% inhibition of B7-2 mRNA in thisassay.

TABLE 11 Nucleotide Sequences of Human B7-2 Chimeric(deoxy gapped) Oligodeoxynucleotides TAGET GENE SEQ NUCLEOTIDE GENE ISISNUCLEOTIDE SEQUENCE¹ ID CO- TARGET NO. (5′->3′) NO: ORDINATES² REGION22284 TGCGAGCTCCCCGTACCTCC  16 0011-0030 5′-UTR 22285CAGAAGCAAGGTGGTAAGAA 175 0049-0068 5′-UTR 22286 GCCTGTCCACTGTAGCTCCA 1760113-0132 5′-UTR 22287 AGAATGTTACTCAGTCCCAT 177 0148-0167 AUG 22288TCAGAGGAGCAGCACCAGAG 178 0189-0208 coding 22289 TGGCATGGCAGGTCTGCAGT 1790232-0251 coding 22290 AGCTCACTCAGGCTTTGGTT 180 0268-0287 coding 22291TGCCTAAGTATACCTCATTC 181 0324-0343 coding 22292 CTGTCAAATTTCTCTTTGCC 1820340-0359 coding 22293 CATATACTTGGAATGAACAC 183 0359-0378 coding 22294GGTCCAACTGTCCGAATCAA 184 0392-0411 coding 22295 TGATCTGAAGATTGTGAAGT 1850417-0436 coding 22296 AAGCCCTTGTCCTTGATCTG 186 0430-0449 coding 22297TGTGATGGATGATACATTGA 187 0453-0472 coding 22298 TCAGGTTGACTGAAGTTAGC 1880529-0548 coding 22299 GTGTATAGATGAGCAGGTCA 189 0593-0612 coding 22300TCTGTGACATTATCTTGAGA 190 0694-0713 coding 22301 AAGATAAAAGCCGCGTCTTG 1910798-0817 coding 22302 AGAAAACCATCACACATATA 192 0900-0919 coding 22303AGAGTTGCGAGGCCGCTTCT 193 0947-0968 coding 22304 TCCCTCTCCATTGTGTTGGT 1940979-0998 coding 22305 CATCAGATCTTTCAGGTATA 195 1035-1054 coding 22306GGCTTTACTCTTTAATTAAA 196 1115-1134 stop 22307 GAAATCAAAAAGGTTGCCCA 1971178-1197 3′-UTR 22308 GGAGTCCTGGAGCCCCCTTA 198 1231-1250 3′-UTR 22309TTGGCATACGGAGCAGAGCT 199 1281-1300 3′-UTR 22310 TGTGCTCTGAAGTGAAAAGA 2001327-1346 3′-UTR 22311 GGCTTGGCCCATAAGTGTGC 201 1342-1361 3′-UTR 22312CCTAAATTTTATTTCCAGGT 202 1379-1398 3′-UTR 24736 GCTCCAAGTGTCCCAATGAA 203control 24737 AGTATGTTTCTCACTCCGAT 204 control 24738TGCCAGCACCCGGTACGTCC 205 control ¹Emboldened residues are2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethylcytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; alllinkages are phosphorothioate linkages. ²Co-ordinates from GenbankAccession No. U04343 locus name “HSU04343”.

TABLE 12 Inhibition of Human B7-2 mRNA Expression byChimeric (deoxy gapped) Phosphorothioate Oligodeoxynucleotides GENE ISISSEQ ID TARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION basal —— 100% 0% 10373  16 5′-UTR 24% 76% 22284  16 5′-UTR 30% 70% 22285 1755′-UTR 74% 26% 22286 176 5′-UTR 39% 61% 22287 177 AUG 27% 73% 22288 178coding 38% 62% 22289 179 coding 41% 59% 22290 180 coding 42% 58% 22291181 coding 41% 59% 22292 182 coding 39% 61% 22293 183 coding 43% 57%22294 184 coding 21% 79% 22295 185 coding 66% 34% 22296 186 coding 42%58% 22297 187 coding 54% 46% 22298 188 coding 53% 47% 22299 189 coding46% 54% 22300 190 coding 39% 61% 22301 191 coding 51% 49% 22302 192coding 41% 59% 22303 193 coding 46% 54% 22304 194 coding 41% 59% 22305195 coding 57% 43% 22306 196 stop 44% 56% 22307 197 3′-UTR 45% 55% 22308198 3′-UTR 40% 60% 22309 199 3′-UTR 42% 58% 22310 200 3′-UTR 41% 59%22311 201 3′-UTR 49% 51% 22312 202 3′-UTR 83% 17%

Dose response experiments were performed on several of the more activeoligonucleotides. The oligonucleotides were screened as described inExample 4 except that the concentration of oligonucleotide was varied asshown in Table 13. Mismatch control oligonucleotides were included.Results are shown in Table 13.

All antisense oligonucleotides tested showed a dose response effect withmaximum inhibition of mRNA approximately 50% or greater.

TABLE 13 Dose Response of COS-7 Cells to B7-2Chimeric (deoxy gapped) Antisense Oligonucleotides SEQ ID ASO Gene% mRNA % mRNA ISIS # NO: Target Dose Expression Inhibition basal — — —100% — 22284  16 5′-UTR  10 nM 92% 8% ″ ″ ″  30 nM 72% 28% ″ ″ ″ 100 nM59% 41% ″ ″ ″ 300 nM 48% 52% 24738 205 control  10 nM 81% 19% ″ ″ ″ 30 nM 92% 8% ″ ″ ″ 100 nM 101% — ″ ″ ″ 300 nM 124% — 22287 177 AUG 10 nM 93% 7% ″ ″ ″  30 nM 79% 21% ″ ″ ″ 100 nM 66% 34% ″ ″ ″ 300 nM 45%55% 24737 204 control  10 nM 85% 15% ″ ″ ″  30 nM 95% 5% ″ ″ ″ 100 nM87% 13% ″ ″ ″ 300 nM 99% 1% 22294 184 coding  10 nM 93% 7% ″ ″ ″  30 nM95% 5% ″ ″ ″ 100 nM 58% 42% ″ ″ ″ 300 nM 45% 55% 24736 203 control 10 nM 102% — ″ ″ ″  30 nM 101% — ″ ″ ″ 100 nM 100% — ″ ″ ″ 300 nM 107%—

Example 15 Chimeric (Deoxy Gapped) Mouse B7-2 Antisense Oligonucleotides

Additional oligonucleotides targeting mouse B7-2 were synthesized.Oligonucleotides were synthesized as uniformly phosphorothioate chimericoligonucleotides having regions of five 2′-O-methoxyethyl (2′-MOE)nucleotides at the wings and a central region of ten deoxynucleotides.Oligonucleotide sequences are shown in Table 14.

Oligonucleotides were screened as described in Example 4. Results areshown in Table 15.

Oligonucleotides 18084 (SEQ ID NO: 206), 18085 (SEQ ID NO: 207), 18086(SEQ ID NO: 208), 18087 (SEQ ID NO: 209), 18089 (SEQ ID NO: 211), 18090(SEQ ID NO: 212), 18091 (SEQ ID NO: 213), 18093 (SEQ ID NO: 215), 18095(SEQ ID NO: 217), 18096 (SEQ ID NO: 218), 18097 (SEQ ID NO: 219), 18098(SEQ ID NO: 108), 18102 (SEQ ID NO: 223), and 18103 (SEQ ID NO: 224)resulted in 50% or greater inhibition of B7-2 mRNA expression in thisassay.

TABLE 14 Nucleotide Sequences of Mouse B7-2 Chimeric(deoxy gapped) Oligodeoxynucleotides TARGET GENE NUCLEOTIDE SEQNUCLEOTIDE GENE ISIS SEQUENCE¹ ID CO- TARGET NO. (5′->3′) NO: ORDINATES²REGION 18084 GCTGCCTACAGGAGCCACTC 206 0003-0022 5′-UTR 18085TCAAGTCCGTGCTGCCTACA 207 0013-0032 5′-UTR 18086 GTCTACAGGAGTCTGGTTGT 2080033-0052 5′-UTR 18087 AGCTTGCGTCTCCACGGAAA 209 0152-0171 coding 18088TCACACTATCAAGTTTCTCT 210 0297-0316 coding 18089 GTCAAAGCTCGTGCGGCCCA 2110329-0348 coding 18090 GTGAAGTCGTAGAGTCCAGT 212 0356-0375 coding 18091GTGACCTTGCTTAGACGTGC 213 0551-0570 coding 18092 CATCTTCTTAGGTTTCGGGT 2140569-0588 coding 18093 GGCTGTTGGAGATACTGAAC 215 0663-0682 coding 18094GGGAATGAAAGAGAGAGGCT 216 0679-0698 coding 18095 ACATACAATGATGAGCAGCA 2170854-0873 coding 18096 GTCTCTCTGTCAGCGTTACT 218 0934-0953 coding 18097TGCCAAGCCCATGGTGCATC 219 0092-0111 AUG 18098 GGATTGCCAAGCCCATGGTG 1080096-0115 AUG 18099 GCAATTTGGGGTTCAAGTTC 220 0967-0986 coding 18100CAATCAGCTGAGAACATTTT 221 1087-1106 3′-UTR 18101 TTTTGTATAAAACAATCATA 2220403-0422 coding 18102 CCTTCACTCTGCATTTGGTT 223 0995-1014 stop 18103TGCATGTTATCACCATACTC 224 0616-0635 coding ¹Emboldened residues are2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethylcytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; alllinkages are phosphorothioate linkages. ²Co-ordinates from GenbankAccession No. S70108 locus name “S70108”.

TABLE 15 Inhibition of Mouse B7-2 mRNA Expression by Chimeric (deoxygapped) Phosphorothioate Oligodeoxynucleotides GENE ISIS TARGET % mRNA %mRNA No: SEQ ID NO: REGION EXPRESSION INHIBITION basal — — 100.0% 0.0%18084 206 5′-UTR 36.4% 63.6% 18085 207 5′-UTR 35.0% 65.0% 18086 2085′-UTR 40.1% 59.9% 18087 209 coding 42.1% 57.9% 18088 210 coding 52.3%47.7% 18089 211 coding 20.9% 79.1% 18090 212 coding 36.6% 63.4% 18091213 coding 37.1% 62.9% 18092 214 coding 58.9% 41.1% 18093 215 coding32.7% 67.3% 18094 216 coding 63.8% 36.2% 18095 217 coding 34.3% 65.7%18096 218 coding 32.3% 67.7% 18097 219 AUG 24.5% 75.5% 18098 108 AUG32.2% 67.8% 18099 220 coding 66.8% 33.2% 18100 221 3′-UTR 67.2% 32.8%18101 222 coding 88.9% 11.1% 18102 223 stop 33.8% 66.2% 18103 224 coding30.2% 69.8%

Example 16 Effect of B7 Antisense Oligonucleotides on Cell SurfaceExpression

B7 antisense oligonucleotides were tested for their effect on cellsurface expression of both B7-1 and B7-2. Cell surface expression wasmeasured as described in Example 1. Experiments were done for both humanB7 and mouse B7. Results for human B7 are shown in Table 16. Results formouse B7 are shown in Table 17.

In both species, B7-1 antisense oligonucleotides were able tospecifically reduce the cell surface expression of B7-1. B7-2 antisenseoligonucleotides were specific for the B-7-2 family member. Theseoligonucleotides were also specific for their effect on B7-1 and B7-2mRNA levels.

TABLE 16 Inhibition of Human B7 Cell Surface Expression by Chimeric(deoxy gapped) Phosphorothioate Oligodeoxynucleotides SEQ ISIS ID GENE %B7-1 % B7-2 No: NO: TARGET EXPRESSION EXPRESSION basal — — 100% 0% 2231626 B7-1 31% 100% 22317 129 B7-1 28% 91% 22320 132 B7-1 37% 86% 22324 135B7-1 37% 91% 22325 136 B7-1 32% 89% 22334 145 B7-1 28% 92% 22335 146B7-1 23% 95% 22337 148 B7-1 48% 101% 22338 36 B7-1 22% 96% 22284 16 B7-288% 32% 22287 177 B7-2 92% 35% 22294 184 B7-2 77% 28%

TABLE 17 Inhibition of Mouse B7 Cell Surface Expression by Chimeric(deoxy gapped) Phosphorothioate Oligodeoxynucleotides SEQ GENE ISIS IDTARGET % B7-1 % B7-2 No: NO: REGION EXPRESSION EXPRESSION basal — — 100%0% 18089 211 B7-2 85% 36% 18097 219 B7-2 87% 28% 18110 161 B7-1 31% 93%18113 164 B7-1 25% 91% 18119 170 B7-1 27% 98%

Dose response experiments were performed on several of the more activehuman B7-1 antisense oligonucleotides. The oligonucleotides werescreened as described in Example 2 except that the concentration ofoligonucleotide was varied as shown in Table 18. Results are shown inTable 18.

All antisense oligonucleotides tested showed a dose response effect withinhibition of cell surface expression approximately 60% or greater.

TABLE 18 Dose Response of COS-7 Cells to B7-1 Chimeric (deoxy gapped)Antisense Oligonucleotides SEQ ID ASO Gene % Surface % Surface ISIS #NO: Target Dose Expression Inhibition basal — — — 100% — 22316 26 5′-UTR10 nM 74% 26% ″ ″ ″ 30 nM 74% 26% ″ ″ ″ 100 nM  47% 53% ″ ″ ″ 300 nM 34% 66% 22335 146  3′-UTR 10 nM 81% 19% ″ ″ ″ 30 nM 69% 31% ″ ″ ″ 100nM  47% 53% ″ ″ ″ 300 nM  38% 62% 22338 36 3′-UTR 10 nM 78% 22% ″ ″ ″ 30nM 65% 35% ″ ″ ″ 100 nM  50% 50% ″ ″ ″ 300 nM  40% 60%

Dose response experiments were performed on several of the more activehuman B7-2 antisense oligonucleotides. The oligonucleotides werescreened as described in Example 2 except that the concentration ofoligonucleotide was varied as shown in Table 19. Results are shown inTable 19.

All antisense oligonucleotides tested showed a dose response effect withmaximum inhibition of cell surface expression 85% or greater.

TABLE 19 Dose Response of COS-7 Cells to B7-2 Chimeric (deoxy gapped)Antisense Oligonucleotides SEQ ID ASO Gene % Surface % Surface ISIS #NO: Target Dose Expression Inhibition basal — — — 100% — 22284  165′-UTR 10 nM 63% 37% ″ ″ ″ 30 nM 60% 40% ″ ″ ″ 100 nM  37% 63% ″ ″ ″ 300nM  15% 85% 22287 177 AUG 10 nM 93% 7% ″ ″ ″ 30 nM 60% 40% ″ ″ ″ 100 nM 32% 68% ″ ″ ″ 300 nM  15% 85% 22294 184 coding 10 nM 89% 11% ″ ″ ″ 30 nM62% 38% ″ ″ ″ 100 nM  29% 71% ″ ″ ″ 300 nM  12% 88%

Example 17 Effect of B7-1 Antisense Oligonucleotides in a Murine Modelfor Rheumatoid Arthritis

Collagen-induced arthritis (CIA) was used as a murine model forarthritis (Mussener, A., et al., Clin. Exp. Immunol., 1997, 107,485-493). Female DBA/1LacJ mice (Jackson Laboratories, Bar Harbor, Me.)between the ages of 6 and 8 weeks were used to assess the activity ofB7-1 antisense oligonucleotides.

On day 0, the mice were immunized at the base of the tail with 100 μg ofbovine type II collagen which is emulsified in Complete Freund'sAdjuvant (CFA). On day 7, a second booster dose of collagen wasadministered by the same route. On day 14, the mice were injectedsubcutaneously with 100 μg of LPS. Oligonucleotide was administeredintraperitoneally daily (10 mg/kg bolus) starting on day −3 (three daysbefore day 0) and continuing for the duration of the study.Oligonucleotide 17456 (SEQ ID NO. 173) is a fully phosphorothioatedanalog of 18122.

Weights were recorded weekly. Mice were inspected daily for the onset ofCIA. Paw widths are rear ankle widths of affected and unaffected jointswere measured three times a week using a constant tension caliper. Limbswere clinically evaluated and graded on a scale from 0-4 (with 4 beingthe highest).

Results are shown in Table 20. Treatment with B7-1 and B7-2 antisenseoligonucleotides was able to reduce the incidence of the disease, buthad modest effects on severity. The combination of 17456 (SEQ ID NO.173) and 11696 (SEQ ID NO. 108) was able to significantly reduce theincidence of the disease and its severity.

TABLE 20 Effect of B7 antisense oligonucleotide on CIA SEQ Dose % ISIS#(s) ID NO mg/kg Inci-dence Peak day¹ Severity² control — 70% 6.7 ± 2.93.2 ± 1.1 17456 (B7-1) 173 10 50% 12.1 ± 4.6  2.7 ± 1.3 11696 (B7-2) 10810 37.5%   11.6 ± 4.5  3.4 ± 1.8 17456/11696 10 30% 1.0 ± 0.6 0.7 ± 0.418110 (B7-1) 161 10 55.6%   2.0 ± 0.8 2.0 ± 1.3 18089 (B7-2) 211 1044.4%   6.8 ± 2.2 2.3 ± 1.3 18110/18089 10 60% 11.6 ± 0.7  4.5 ± 1.7¹Peak day is the day from onset of maximum swelling for each jointmeasure. ²Severity is the total clinical score divided by the totalnumber of mice in the group.

Example 18 Effect of B7-1 Antisense Oligonucleotides in a Murine Modelfor Multiple Sclerosis

Experimental autoimmune encephalomyelitis (EAE) is a commonly acceptedmurine model for multiple sclerosis (Myers, K. J., et al., J.Neuroimmunol., 1992, 41, 1-8). SJL/H, PL/J, (SJLxPL/J)F1, (SJLxBalb/c)F1and Balb/c female mice between the ages of 6 and 12 weeks are used totest the activity of a B7-1 antisense oligonucleotide.

The mice are immunized in the two rear foot pads and base of the tailwith an emulsion consisting of encephalitogenic protein or peptide(according to Myers, K. J., et al., J. of Immunol., 1993, 151,2252-2260) in Complete Freund's Adjuvant supplemented with heat killedMycobacterium tuberculosis. Two days later, the mice receive anintravenous injection of 500 ng Bordetella pertussis toxin andadditional adjuvant.

Alternatively, the disease may also be induced by the adoptive transferof T-cells. T-cells are obtained from the draining of the lymph nodes ofmice immunized with encephalitogenic protein or peptide in CFA. The Tcells are grown in tissue culture for several days and then injectedintravenously into naive syngeneic recipients.

Mice are monitored and scored daily on a 0-5 scale for signals of thedisease, including loss of tail muscle tone, wobbly gait, and variousdegrees of paralysis.

Oligonucleotide 17456 (SEQ ID NO. 173), a fully phosphorothioated analogof 18122, was compared to a saline control and a fully phosphorothioatedoligonucleotide of random sequence (Oligonucleotide 17460). Results ofthis experiment are shown in FIG. 11.

As shown in FIG. 11, for all doses of oligonucleotide 17456 tested,there is a protective effect, i.e. a reduction of disease severity. At0.2 mg/kg, this protective effect is greatly reduced after day 20, butat the higher doses tested, the protective effect remains throughout thecourse of the experiment (day 40). The control oligonucleotide gaveresults similar to that obtained with the saline control.

Example 19 Additional Antisense Oligonucleotides Targeted to Human B7-1

Additional oligonucleotides targeting human B7-1 were synthesized.Oligonucleotides were synthesized as uniformly phosphorothioate chimericoligonucleotides having regions of five 2′-O-methoxyethyl (2′-MOE)nucleotides at the wings and a central region often deoxynucleotides.Oligonucleotide sequences are shown in Table 21.

The human promonocytic leukaemia cell line, THP-1 (American Type CultureCollection, Manassas, Va.) was maintained in RPMI 1640 growth mediasupplemented with 10% fetal calf serum (FCS; Life Technologies,Rockville, Md.). A total of 1×10⁷ cells were electroporated at anoligonucleotide concentration of 10 micromolar in 2 mm cuvettes, usingan Electrocell Manipulator 600 instrument (Biotechnologies andExperimental Research, Inc.) employing 200 V, 1000 μF. Electroporatedcells were then transferred to petri dishes and allowed to recover for16 hrs. Cells were then induced with LPS at a final concentration of 1μg/ml for 16 hours. RNA was isolated and processed as described inprevious examples. Results are shown in Table 22.

Oligonucleotides 113492, 113495, 113498, 113499, 113501, 113502, 113504,113505, 1113507, 113510, 113511, 113513 and 113514 (SEQ ID NO: 228, 231,234, 235, 237, 238, 240, 241, 243, 246, 247, 249 and 250) resulted in50% or greater inhibition of B7-1 mRNA expression in this assay.

TABLE 21 Nucleotide Sequences of Human B7-1 Chimeric(deoxy gapped) Oligodeoxynucleotides NUCLEOTIDE SEQ TARGET GENE GENEISIS SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′->3′) NO. CO-ORDINATES²REGION 113489 CCCTCCAGTGATGTTTACAA 225 179 5′UTR 113490GAAGACCCTCCAGTGATGTT 226 184 5′UTR 113491 CGTAGAAGACCCTCCAGTGA 227 1885′UTR 113492 TTCCCAGGTGCAAAACAGGC 228 234 5′UTR 113493TGGCTTCAGATGCTTAGGGT 229 299 5′UTR 113494 CCTCCGTGTGTGGCCCATGG 230 316AUG 113495 GGTGATGTTCCCTGCCTCCG 231 330 Coding 113496GATGGTGATGTTCCCTGCCT 232 333 Coding 113497 AGGTATGGACACTTGGATGG 233 348Coding 113498 GAAAGACCAGCCAGCACCAA 234 384 Coding 113499CAGCGTTGCCACTTCTTTCA 235 442 Coding 113500 GTGACCACAGGACAGCGTTG 236 454Coding 113501 AGATGCGAGTTTGTGCCAGC 237 491 Coding 113502CCTTTTGCCAGTAGATGCGA 238 503 Coding 113503 CGGTTCTTGTACTCGGGCCA 239 567Coding 113504 CGCAGAGCCAGGATCACAAT 240 618 Coding 113505CTTCAGCCAGGTGTTCCCGC 241 698 Coding 113506 TAACGTCACTTCAGCCAGGT 242 706Coding 113507 TTCTCCATTTTCCAACCAGG 243 838 Coding 113508CTGTTGTGTTGATGGCATTT 244 863 Coding 113509 CATGAAGCTGTGGTTGGTTG 245 943Coding 113510 AGGAAAATGCTCTTGCTTGG 246 1018 Coding 113511TGGGAGCAGGTTATCAGGAA 247 1033 Coding 113512 TAAGGTAATGGCCCAGGATG 2481051 Coding 113513 GGTCAGGCAGCATATCACAA 249 1090 Coding 113514GCCCCTTGCTTCTGCGGACA 250 1188 3′UTR 113515 AGATCTTTTCAGCCCCTTGC 251 11993′UTR 113516 TTTGTTAAGGGAAGAATGCC 252 1271 3′UTR 113517AAAGGAGAGGGATGCCAGCC 253 1362 3′UTR 113518 CAAGACAATTCAAGATGGCA 254 14363′UTR ¹Emboldened residues are 2′-methoxyethoxy residues (others are2′-deoxy-). All 2′-methoxyethyl cytosines and 2′-deoxy cytosinesresidues are 5-methyl-cytosines; all linkages are phosphorothioatelinkages. ²Co-ordinates from Genbank Accession No. M27533 to which theoligonucleotides are targeted.

TABLE 22 Inhibition of Human B7-1 mRNA Expression by Chimeric (deoxygapped) Phosphorothioate Oligodeoxynucleotides GENE ISIS SEQ ID TARGET %mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION 113489 225 5′ UTR 122 —113490 226 5′ UTR 183 — 113491 227 5′ UTR 179 — 113492 228 5′ UTR 27 73113493 229 5′ UTR 488 — 113494 230 AUG 77 23 113495 231 Coding 43 57113496 232 Coding 71 29 113497 233 Coding 78 22 113498 234 Coding 37 63113499 235 Coding 25 75 113500 236 Coding 83 17 113501 237 Coding 36 64113502 238 Coding 26 74 113503 239 Coding 65 35 113504 240 Coding 46 54113505 241 Coding 40 60 113506 242 Coding 105 — 113507 243 Coding 36 64113508 244 Coding 117 — 113509 245 Coding 62 38 113510 246 Coding 43 57113511 247 Coding 48 52 113512 248 Coding 73 27 113513 249 Coding 48 52113514 250 3′ UTR 35 65 113515 251 3′ UTR 184 — 113516 252 3′ UTR 83 17113517 253 3′ UTR 201 — 113518 254 3′ UTR 97 03

Example 20 Additional Antisense Oligonucleotides Targeted to Human B7-2

Additional oligonucleotides targeting human B7-2 were synthesized.Oligonucleotides were synthesized as uniformly phosphorothioate chimericoligonucleotides having regions of five 2′-O-methoxyethyl (2′-MOE)nucleotides at the wings and a central region of ten deoxynucleotides.Oligonucleotide sequences are shown in Table 23.

The human promonocytic leukaemia cell line, THP-1 (American Type CultureCollection, Manassas, Va.) was maintained in RPMI 1640 growth mediasupplemented with 10% fetal calf serum (FCS; Life Technologies,Rockville, Md.). A total of 1×10⁷ cells were electroporated at anoligonucleotide concentration of 10 micromolar in 2 mm cuvettes, usingan Electrocell Manipulator 600 instrument (Biotechnologies andExperimental Research, Inc.) employing 200 V, 1000 μF. Electroporatedcells were then transferred to petri dishes and allowed to recover for16 hrs Cells were then induced with LPS and dibutyryl cAMP (500 μM) for16 hours. RNA was isolated and processed as described in previousexamples. Results are shown in Table 24.

Oligonucleotides ISIS 113131, 113132, 113134, 113138, 113142, 113144,113145, 113146, 113147, 113148, 113149, 113150, 113153, 113155, 113157,113158, 113159 and 113160 (SEQ ID NO: 255, 256, 258, 262, 266, 268, 269,270, 271, 272, 273, 274, 277, 279, 281, 282, 283 and 284) resulted in50% or greater inhibition of B7-2 mRNA expression in this assay.

TABLE 23 Nucleotide Sequences of Human B7-2 Chimeric(deoxy gapped) Oligodeoxynucleotides SEQ TARGET GENE GENE ISISNUCLEOTIDE SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′->3′) NO: CO-ORDINATES²REGION 113131 CGTGTGTCTGTGCTAGTCCC 255 38 5′UTR 113132GCTGCTTCTGCTGTGACCTA 256 83 5′UTR 113133 TATTTGCGAGCTCCCCGTAC 257 155′UTR 113134 GCATAAGCACAGCAGCATTC 258 79 5′UTR 113135TCCAAAAAGAGACCAGATGC 259 97 5′UTR 113136 AAATGCCTGTCCACTGTAGC 260 1175′UTR 113137 CTTCAGAGGAGCAGCACCAG 261 191 Coding 113138GAATCTTCAGAGGAGCAGCA 262 195 Coding 113139 CAAATTGGCATGGCAGGTCT 263 237Coding 113140 GCTTTGGTTTTGAGAGTTTG 264 257 Coding 113141AGGCTTTGGTTTTGAGAGTT 265 259 Coding 113142 GCTCACTCAGGCTTTGGTTT 266 267Coding 113143 GGTCCTGCCAAAATACTACT 267 288 Coding 113144AGCCCTTGTCCTTGATCTGA 268 429 Coding 113145 TGTGGGCTTTTTGTGATGGA 269 464Coding 113146 AATCATTCCTGTGGGCTTTT 270 473 Coding 113147CCGTGTATAGATGAGCAGGT 271 595 Coding 113148 ACCGTGTATAGATGAGCAGG 272 596Coding 113149 TCATCTTCTTAGGTTCTGGG 273 618 Coding 113150ACAAGCTGATGGAAACGTCG 274 720 Coding 113151 TGCTCGTAACATCAGGGAAT 275 747Coding 113152 AAGATGGTCATATTGCTCGT 276 760 Coding 113153CGCGTCTTGTCAGTTTCCAG 277 787 Coding 113154 CAGCTGTAATCCAAGGAATG 278 864Coding 113155 GGGCTTCATCAGATCTTTCA 279 1041 Coding 113156CATGTATCACTTTTGTCGCA 280 1093 Coding 113157 AGCCCCCTTATTACTCATGG 2811221 3′UTR 113158 GGAGTTACAGGGAGGCTATT 282 1261 3′UTR 113159AGTCTCCTCTTGGCATACGG 283 1290 3′UTR 113160 CCCATAAGTGTGCTCTGAAG 284 13353′UTR ¹Emboldened residues are 2′-methoxyethoxy residues (others are2′-deoxy-). All 2′-methoxyethyl cytosines and 2′-deoxy cytosinesresidues are 5-methyl-cytosines; all linkages are phosphorothioatelinkages. ²For ISIS #113131 and 113132, co-ordinates are from GenbankAccession No. L25259, locus name “HUMB72A”. For remainingoigonucleotides, co-ordinates are from Genbank Accession No. U04343,locus name “HSUU04343”.

TABLE 24 Inhibition of Human B7-2 mRNA Expression byChimeric (deoxy gapped) Phosphorothioate Oligodeoxynucleotides GENE ISISSEQ ID TARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION 113131255 5′UTR 13 87 113132 256 5′UTR 17 83 113133 257 5′UTR 214 — 113134 2585′UTR 27 73 113135 259 5′UTR 66 34 113136 260 5′UTR 81 19 113137 261Coding 57 43 113138 262 Coding 12 88 113140 264 Coding 214 — 113141 265Coding 126 — 113142 266 Coding 35 65 113143 267 Coding 118 — 113144 268Coding 41 59 113145 269 Coding 46 54 113146 270 Coding 32 68 113147 271Coding 35 65 113148 272 Coding 23 77 113149 273 Coding 29 71 113150 274Coding 19 81 113151 275 Coding 208 — 113152 276 Coding 89 11 113153 277Coding 19 81 113154 278 Coding 63 37 113155 279 Coding 13 87 113156 280Coding 83 17 113157 281 3tUTR 13 87 113158 282 3′UTR 20 80 113159 2833′UTR 43 57 113160 284 3′UTR 09 91

Example 21 Human Skin Psoriasis Model

Animal models of psoriasis based on xenotransplantation of human skinfrom psoriatic patients are advantageous because they involve the directstudy of affected human tissue. Psoriasis is solely a disease of theskin and consequently, engraftment of human psoriatic skin onto SCIDmice allows psoriasis to be created with a high degree of fidelity inmice.

BALB/cByJSmn-Prkdcscid/J SCID mice (4-6 weeks old) of either sex(Jackson Laboratory, Bar Harbor, Me.) were maintained in a pathogen freeenvironment. At 6-8 weeks of age, mice were anesthetized byintraperitoneal injection of 30 mg/kg body weight ketamine-HCl and 1mg/kg body weight acepromazine. After anesthesia, mice were prepared fortransplantation by shaving the hair from the dorsal skin, 2 cm away fromthe head. The area was then sterilized and cleaned with povidone iodideand alcohol. Graft beds of about 1 cm×1 cm were created on the shavedareas by removing full thickness skin down to the fascia. Partialthickness human skin was then orthotopically transferred onto the graftbed. The transplants were held in place by gluing the human skin tomouse-to-mouse skin with Nexband liquid, a veterinary bandage(Veterinary Products Laboratories, Phoenix, Ariz.). Finally, thetransplant and the wounds were covered with a thick layer of antibioticointment. After 4 weeks of transplantation, a 2 mm punch biopsy wasobtained to confirm the acceptance of the graft and the origin of theskin in the transplant area. Only mice whose grafts did not show signsof infection were used for the study. Normal human skin was obtainedfrom elective plastic surgeries and psoriatic plaques were obtained fromshave biopsies from psoriatic volunteers. Partial thickness skin wasprepared by dermatome shaving of the skin and transplanted to the mouseas described above for the psoriatic skin.

Animals (n=5) were topically treated with 2.5% (w/w) of each antisenseoligonucleotide in a cream formulation comprising 10% isopropylmyristate, 10% glyceryl monooleate, 3% cetostearyl alcohol, 10%polyoxyl-20-cetyl ether, 6% poloxamer 407, 2.5% phenoxyethanol, 0.5%methylparaben, 0.5% propylparaben and water (final pH about 7.5).

The following oligonucleotides were used: human B7-1(5′-TTCCCAGGTGCAAAACAGGC-3′; SEQ ID NO: 228) (ISIS 113492) and humanB7-2 (5′-CGTGTGTCTGTGCTAGTCCC-3′; SEQ ID NO: 255) (ISIS 113131). Bothsequences contained only phosphorothioate linkages and had 2′-MOEmodifications at nucleotides 1-5 and 16-20.

Plaques from the same patients were also transplanted onto control mice(n=5) and treated only with the vehicle of the active cream preparation.Both groups received the topical preparation twice a day for 4 weeks.Within 3-4 weeks the animals were sacrificed and 4 mm punch biopsieswere taken from each xenograft. Biopsies were fixed in formalin forparaffin embedding and/or transferred to cryotubes and snap-frozen inliquid nitrogen and stored at −80° C.

Significant histological improvement marked by reduction ofhyperkeratosis, acanthosis and lymphonuclear cellular infiltrates wasobserved in mice treated with the antisense oligonucleotides. Rete pegs,finger-like projections of the epidermis into the dermis, were alsomeasured. These are phenotypic markers for psoriasis which lengthen asthe disease progresses. The shortening of these rete pegs is a goodmeasure of anti-psoriatic activity. In mice treated with the activeagent, the rete pegs changed from 238.56±98.3 μm to 168.4±96.62 μm(p<0.05), whereas in the control group the rete pegs before and aftertreatment were 279.93±40.56 μm and 294.65±45.64 μm, respectively(p>0.1). HLA-DR positive lymphocytic infiltrates and intraepidermal CD8positive lymphocytes were significantly reduced in the transplantedplaques treated with the antisense oligonucleotide cream. These resultsshow that antisense oligonucleotides to B7 inhibit psoriasis-inducedinflammation and have therapeutic efficacy in the treatment ofpsoriasis.

Example 22 Mouse Model of Allergic Inflammation

In the mouse model of allergic inflammation, mice were sensitized andchallenged with aerosolized chicken ovalbumin (OVA). Airwayresponsiveness was assessed by inducing airflow obstruction with amethacholine aerosol using a noninvasive method. This methodologyutilized unrestrained conscious mice that are placed into the mainchamber of a plthysmograph (Buxco Electronics, Inc., Troy, N.Y.).Pressure differences between this chamber and a reference chamber wereused to extrapolate minute volume, breathing frequency and enhancedpause (Penh). Penh is a dimensionless parameter that is a function oftotal pulmonary airflow in mice (i.e., the sum of the airflow in theupper and lower respiratory tracts) during the respiratory cycle of theanimal. The lower the Penh, the greater the airflow. This parameterclosely correlates with lung resistance as measured by traditionalinvasive techniques using ventilated animals (Hamelmann . . . Gelfand,1997). Dose-response data were plotted as raw Penh values to increasingconcentrations of methacholine. This system was used to test theefficacy of antisense oligonucleotides targeted to human B7-1 and B7-2.

There are several important features common to human asthma and themouse model of allergic inflammation. One of these is pulmonaryinflammation, in which cytokine expression and Th2 profile is dominant.Another is goblet cell hyperplasia with increased mucus production.Lastly, airway hyperresponsiveness (AHR) occurs resulting in increasedsensitivity to cholinergic receptor agonists such as acetylcholine ormethacholine. The compositions and methods of the present invention maybe used to treat AHR and pulmonary inflammation.

Ovalbumin-Induced Allergic Inflammation

Female Balb/c mice (Charles Rivers Laboratory, Taconic Farms, N.Y.) weremaintained in micro-isolator cages housed in a specific pathogen-free(SPF) facility. The sentinel cages within the animal colony surveyednegative for viral antibodies and the presence of known mouse pathogens.Mice were sensitized and challenged with aerosolized chicken OVA.Briefly, 20 μg alum-precipitated OVA was injected intraperitoneally ondays 0 and 14. On day 24, 25 and 26, the animals were exposed for 20minutes to 1.0% OVA (in saline) by nebulization. The challenge wasconducted using an ultrasonic nebulizer (PulmoSonic, The DeVilbiss Co.,Somerset, Pa.). Animals were analyzed about 24 hours following the lastnebulization using the Buxco electronics Biosystem. Lung function(Penh), lung histology (cell infiltration and mucus production), targetmRNA reduction in the lung, inflammation (BAL cell type & number,cytokine levels), spleen weight and serum AST/ALT were determined.

This method has been used to show that prophylactic treatment with ananti-B7.2 monoclonal antibody continued throughout the sensitization andchallenge periods decreases OVA-specific serum IgE and IgE levels, IL-4and IFN-γ levels in bronchoalveolar lavage (BAL) fluid, airwayeosinophilia and airway hyperresponsiveness (Haczku et al., Am. J.Respir. Crit. Care Med. 159:1638-1643, 1999). Treatment during antigenchallenge with both anti-B7.1 and anti-B7.2 mAbs is effective; however,either mAb alone is only partially active (Mathur et al., 21:498-509,1999). However, the anti-B7.2 mAb had no activity when administeredafter the OVA challenge. The anti-B7.1 monoclonal antibody had noeffect, either prophylactically or post-antigen challenge. Thus, thereis a need for an effective B7 inhibitor which can be administered afterantigen challenge, and which will reduce airway hyperresponsiveness andpulmonary inflammation. As described below, the antisenseoligonucleotides of the present inventors fit this description.

Oligonucleotide Administration

Antisense oligonucleotides (ASOs) were dissolved in saline and used tointratracheally dose mice every day, four times per day, from days 15-26of the OVA sensitization and challenge protocol. Specifically, the micewere anesthetized with isofluorane and placed on a board with the frontteeth hung from a line. The nose was covered and the animal's tongue wasextended with forceps and 251 μl of various doses of ASO, or anequivalent volume of saline (control) was placed at the back of thetongue until inhaled into the lung. The deposition pattern of an ASO inthe lung, ISIS 13920 (5′-TCCGTCATCGCTCCTCAGGG-3′; SEQ ID NO:285) wasalso examined by immunohistochemical staining using a monoclonalantibody to the oligonucleotide, and showed that the ASO is taken upthroughout the lung, most strongly by antigen presenting cells (APCs)and alveolar epithelium.

The B7 oligonucleotides used were:

B7-1: 5′-GCTCAGCCTTTCCACTTCAG-3′ (ISIS 121844; SEQ ID NO: 286) B7-2:5′-GCTCAGCCTTTCCACTTCAG-3′ (ISIS 121874; SEQ ID NO: 287)

Both of these oligonucleotides are phosphorothioates with 2′-MOEmodifications on nucleotides 1-5 and 16-20, and 2′-deoxy at positions6-15. These ASOs were identified by mouse-targeted ASO screening bytarget mRNA reduction in mouse cell lines. For B7-2, 19 mouse-targetedASOs were screened by target mRNA reduction (Northern analysis) in IC-21macrophages. Dose-response confirmation led to selection of ISIS 121874(>70% reduction at 25 nM). For B7-1, 22 mouse-targeted ASOs werescreened by target mRNA reduction (RT-PCR) in L-929 fibroblasts.Dose-response confirmation led to selection of ISIS 121844 (>70%reduction at 100 nM). No cross hybridization was predicted, and nocross-target reduction was detected in transfected cells.

RT-PCR Analysis

RNA was harvested from experimental lungs removed on day 28 of the OVAprotocol. B7.2 and B7.1 levels were measured by quantitative RT-PCRusing the Applied Biosystems PRISM 7700 Sequence Detection System(Applied Biosystems, Foster City, Calif.). Primers and probes used forthese studies were synthesized by Operon Technologies (Alameda, Calif.).The primer and probe sequences were as follows:

B7-2: (SEQ ID NO: 288) forward: 5′-GGCCCTCCTCCTTGTGATG-3′(SEQ ID NO: 289) probe: 5′-/56-FAM/TGCTCATCATTGTATGTCACAAGAAGCCG/36-TAMTph/-3′ (SEQ ID NO: 290) reverse: 5′-CTGGGCCTGCTAGGCTGAT-3′ B7-1:(SEQ ID NO: 291) forward: 5′-CAGGAAGCTACGGGCAAGTT-3′ (SEQ ID NO: 292)probe: 5′-/56-FAM/TGGGCCTTTGATTGCTTGATGACTGAA/36- TAMTph/-3′(SEQ ID NO: 293) reverse: 5′-GTGGGCTCAGCCTTTCCA-3′Collection of Bronchial Alveolar Lavage (BAL) Fluid and Blood Serum forthe Determination of Cytokine and Chemokine Levels

Animals were injected with a lethal dose of ketamine, the trachea wasexposed and a cannula was inserted and secured by sutures. The lungswere lavaged twice with 0.5 ml aliquots of ice cold PBS with 0.2% FCS.The recovered BAL fluid was centrifuged at 1,000 rpm for 10 min at 4°C., frozen on dry ice and stored at −80° C. until used. Luminex was usedto measure cytokine levels in BAL fluid and serum.

BAL Cell Counts and Differentials

Cytospins of cells recovered from BAL fluid were prepared using aShandon Cytospin 3 (Shandon Scientific LTD, Cheshire, England). Celldifferentials were performed from slides stained with Leukostat (FisherScientific, Pittsburgh, Pa.). Total cell counts were quantified byhemocytometer and, together with the percent type by differential, wereused to calculate specific cell number.

Tissue Histology

Before resection, lungs were inflated with 0.5 ml of 10%phosphate-buffered formalin and fixed overnight at 4° C. The lungsamples were washed free of formalin with 1×PBS and subsequentlydehydrated through an ethanol series prior to equilibration in xyleneand embedded in paraffin. Sections (6μ) were mounted on slides andstained with hematoxylin/eosin, massons trichome and periodicacid-schiff (PAS) reagent. Parasagittal sections were analyzed bybright-field microscopy. Mucus cell content was assessed as the airwayepithelium staining with PAS. Relative comparisons of mucus content weremade between cohorts of animals by counting the number of PAS-positiveairways.

As shown in FIGS. 11A-11B, B7.2 mRNA (FIG. 11A) and B7.1 mRNA (FIG. 11B)were detected in mouse lung and lymph node during the development ofovalbumin-induced asthma. Treatment with ISIS 121874 following allergenchallenge reduces the airway response to methacholine (FIG. 12). ThePenh value in B7.2 ASO-treated mice was about 40% lower thanvehicle-treated mice, and was statistically the same as naïve mice whichwere not sensitized with the allergen or treated with the ASO. Thisshows that B7.2 ASO-treated mice had significantly better airflow, andless inflammation, than mice which were not treated with the ASO. Thedose-dependent inhibition of the Penh response to methacholine by ISIS121874 is shown in FIG. 13. The inhibition of allergen-inducedeosinophilia by ISIS 121874 is shown in FIG. 14. ISIS 121874 at 0.3mg/kg reduced the total number of eosinophils by about 75% compared tovehicle-treated mice. Since increased numbers of eosinophils result frominflammation, this provides further support for the anti-inflammatoryproperties of the B7.2 ASO. In addition, daily intratracheal delivery ofISIS 121874 does not induce splenomegaly, the concentration of ISIS121874 achieved in lung tissue via daily intratracheal administration isproportional to the dose delivered (FIG. 15) and ISIS 121874 is retainedin lung tissue for at least one week following single dose (0.3 mg/kg)intratracheal administration as determined by capillary gelelectrophoresis (CGE) analysis (FIG. 16).

Example 23 Support for an Antisense Mechanism of Action for ISIS 121874

Two variants of ISIS 121874 were synthesized: a 7 base mismatch5′-TCAAGTCCTTCCACACCCAA-3′ (ISIS 306058; SEQ ID NO: 294) and a gapablated oligonucleotide ISIS 306058 having the same sequence as ISIS121874, but with 2′-MOE modifications at nucleotides 1, 2, 3, 6, 9, 13,16, 18, 19 and 20. Because of the presence of 2′-MOE in the gap, thisoligonucleotide is no longer an RNase H substrate and will not recruitRNase H to the RNA-DNA hybrid which is formed.

The results (FIG. 17) show that at 0.3 mg/kg, only ISIS 121874, and notthe mismatch and gap ablated controls, significantly lowered Penh, whichsupports that ISIS 121874 is working by an antisense mechanism.

The effects of ISIS 121874 and the control oligonucleotides on airwaymucus production in the ovalbumin-induced model were also tested. Theresults (FIG. 18) show that only ISIS 121874 significantly inhibitedmucus production.

The effect of ISIS 121874 on B7.2 and B7.1 mRNA in lung tissue ofallergen-challenged mice is shown in FIGS. 19A and 19B, respectively.The effect of ISIS 121874 on B7.2 and B7.1 mRNA in draining lymph nodesof allergen-challenged mice is shown in FIGS. 20A and 20B, respectively.This shows that ISIS 121874 reduces both B7.2 and B7.1 mRNA (greater inlung vs. node).

In summary, ISIS 121874 resulted in a dose-dependent inhibition ofairway hypersensitivity, inhibited eosinophilia and reduced B7.1 andB7.2 expression in the lung and lymph nodes. In addition, ISIS 121874reduced levels of the following inflammatory molecules: IgE mRNA in thelung and IgE protein in the serum; reduced IL-5 mRNA in the lung andIL-5 protein in the BAL fluid; and reduced the serum level of macrophagechemokine (KC).

In the aerosolized ISIS 121874 study, treatment with 0.001, 0.01, 0.1 or1.0 mg/kg estimated inhaled dose was delivered by nose-only inhalationof an aerosol solution, four times per day, on days 15-26 (n=8 mice pergroup). The airway response to methacholine was reduced to the levelseen in naïve mice at 0.001 mg/kg dose (estimated inhaled dose=1 μg/kg).No gross adverse effects were seen.

Example 24 B7.1 ASO in Ovalbumin Model of Asthma

The same protocols described above for the B7.2 ASOs were used to testthe effect of the B7.1 ASO ISIS 121844 (SEQ ID NO: 286). In contrast tothe B7.2 ASO, ISIS 121844 had no effect on the Penh response in micechallenged with methacholine. Although there was no effect on Penh, ISIS121844 reduced allergen-induced airway eosinophilia (FIG. 21) andreduced the levels of B7.1 and B7.2 in the mouse lung. (FIGS. 22A-B).Thus, treatment with B7.1 ASO produced anti-inflammatory effects, butdid not prevent airway hyper-responsiveness. There was no effect of ISIS121844 on the Penh response despite achieving an 80% reduction of B7.2mRNA in the lung (FIG. 21B). Treatment with ISIS 121844 reducedeosinophil and PMN numbers in BAL fluid. This effect was associated witha reduction in lung B7.2 (not B7.1) mRNA.

The combined use of B7.1 or B7.2 with one or more conventional asthmamedications including, but not limited to, montelukast sodium(Singulair™), albuterol, beclomethasone dipropionate, triamcinoloneacetonide, ipratropium bromide (Atrovent™), flunisolide, fluticasonepropionate (Flovent™) and other steroids is also contemplated. Thecombined use of oligonucleotides which target both B7.1 and B7.2 for thetreatment of asthma is also within the scope of the present invention.B7.1 and B7.2 may also be combined with one or more conventional asthmamedications as described above for B7.1 or B7.2 alone.

Example 25 Design and Screening of Duplexed Antisense CompoundsTargeting B7.1 or B7.2

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements can be designed to target B7.1 or B7.2. The nucleobasesequence of the antisense strand of the duplex comprises at least aportion of an oligonucleotide to B7.1 or B7.2 as described herein. Theends of the strands may be modified by the addition of one or morenatural or modified nucleobases to form an overhang. The sense strand ofthe dsRNA is then designed and synthesized as the complement of theantisense strand and may also contain modifications or additions toeither terminus. For example, in one embodiment, both strands of thedsRNA duplex would be complementary over the central nucleobases, eachhaving overhangs at one or both termini. For example, a duplexcomprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG(SEQ ID NO: 445) and having a two-nucleobase overhang ofdeoxythymidine(dT) would have the following structure:

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 μM. Once diluted, 30μL of each strand is combined with 15 μL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 μL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 μM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate B7.1 or B7.2 expression according to the protocolsdescribed herein.

Example 26 Design of Phenotypic Assays and In Vivo Studies for The Useof B7.1 or B7.2 Inhibitors

Phenotypic Assays

Once B7.1 or B7.2 inhibitors have been identified by the methodsdisclosed herein, the compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of B7.1 or B7.2 in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.), cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with B7.1 orB7.2 inhibitors identified from the in vitro studies as well as controlcompounds at optimal concentrations which are determined by the methodsdescribed above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the genotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the B7.1 or B7.2 inhibitors.Hallmark genes, or those genes suspected to be associated with aspecific disease state, condition, or phenotype, are measured in bothtreated and untreated cells.

Example 27 Antisense Inhibition of Human B7.2 Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, an additional series ofantisense compounds were designed to target different regions of thehuman B7.2 RNA, using published sequences (GenBank accession numberU04343.1, incorporated herein as SEQ ID NO: 295, GenBank accessionnumber BC040261.1, incorporated herein as SEQ ID NO: 296 and GenBankaccession number NT_(—)005543.12, a portion of which is incorporatedherein as SEQ ID NO: 297). The compounds are shown in Table 25. “Targetsite” indicates the first (5′-most) nucleotide number on the particulartarget sequence to which the compound binds. All compounds in Table 25are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length,composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human B7.2 mRNA levels in THP-1 cells by quantitativereal-time PCR as described in other examples herein. Data are averagesfrom three experiments. If present, “N.D.” indicates “no data”.

TABLE 25 Inhibition of human B7.2 mRNA levels by chimeric phos-phorothioate oligonucleotides having 2′-MOE wings and a deoxy gap SEQGenbank Isis Sequence ID Accesion Target Number 5′ to 3′ NO: % INHIBTarget Site Region 322216 ACCAAAAGGAGTATTTGCGA 298 N.D. U04343.1 265′UTR 322217 CATTCCCAAGGAACACAGAA 299 N.D. U04343.1 64 5′UTR 322218ACTGTAGCTCCAAAAAGAGA 300 N.D. U04343.1 105 5′UTR 322219CTGTCACAAATGCCTGTCCA 301 N.D. U04343.1 124 5′UTR 322220TCAGTCCCATAGTGCTGTCA 302 N.D. U04343.1 138 START 322221CTGTTACAGCAGCAGAGAAG 303 N.D. BC040261.1 29 5′UTR 322222TCCCTGTTACAGCAGCACTC 304 N.D. BC040261.1 32 5′UTR 322223ATCTGGAAATGACCCCAAAT 305 N.D. BC040261.1 71 5′UTR 322224GTGACCTAATATCTGGACTG 306 N.D. BC040261.1 81 5′UTR 322225CATTTTGGCTGCTTCTGCTG 307 N.D. BC040261.1 100 START 322226GGAACTTACAAAGGAAAGGG 308 N.D. BC040261.1 1145 3′UTR 322227AAAAAGGTTGCCCAGGAACT 309 N.D. BC040261.1 1159 3′UTR 322228TGCCTTCTGGAAGAAATCAA 310 N.D. BC040261.1 1177 3′UTR 322229TTTTTGCCTTCTGGAAGAAA 311 N.D. BC040261.1 1181 3′UTR 322230CTATTCCACTTAGAGGGAGT 312 N.D. BC040261.1 1233 3′UTR 322231TCTGATCTGGAGGAGGTATT 313 N.D. BC040261.1 1389 3′UTR 322232AGAAATTGAGAGGTCTATTT 314 N.D. BC040261.1 1444 3′UTR 322233CACCAGCTTAGAATTCTGGG 315 N.D. BC040261.1 1484 3′UTR 322234AGGTAGTTGTTTAGTCACAG 316 N.D. BC040261.1 1524 3′UTR 322235CCAGACTGAGGAGGTAGTTG 317 N.D. BC040261.1 1535 3′UTR 322236CAGTACATAGATCTCTATGT 318 N.D. BC040261.1 1599 3′UTR 322237TTACAGTACATAGATCTCTA 319 N.D. BC040261.1 1602 3′UTR 322238GATGAGAACTCCTTAGCAGG 320 N.D. BC040261.1 1657 3′UTR 322239TAGCAACAGCCCAGATAGAA 321 N.D. BC040261.1 1787 3′UTR 322240TCTGTTGCTTGTTTCAAGAC 322 N.D. BC040261.1 2043 3′UTR 322241TCCATTTGGACAGACTATCC 323 N.D. BC040261.1 2064 3′UTR 322242GGGAAACTGCTGTCTGTCTT 324 N.D. BC040261.1 2087 3′UTR 322243TGCTTCCAGGAAGATGACAT 325 N.D. BC040261.1 2149 3′UTR 322244ATTCATCCCATTATCAAGGT 326 N.D. BC040261.1 2191 3′UTR 322245AGCCAGGAGTGGAAAGTCCT 327 N.D. BC040261.1 2223 3′UTR 322246CTTCCTAATTCCGTTGCAGC 328 N.D. BC040261.1 2255 3′UTR 322247CATCTGTAGGCTAAGTAAGC 329 N.D. BC040261.1 2297 3′UTR 322248CCCGTAGGACATCTGTAGGC 330 N.D. BC040261.1 2306 3′UTR 322249GCCCTATGCTGGGCCAGCCC 331 N.D. BC040261.1 2331 3′UTR 322250GTCTCTGTATGCAAGTTTCC 332 N.D. BC040261.1 2396 3′UTR 322251CCAGTATATCTGTCTCTGTA 333 N.D. BC040261.1 2407 3′UTR 322252CCAGGTTTTCAAAGTCATTT 334 N.D. BC040261.1 2430 3′UTR 322253AGCCAGGTTTTCAAAGTCAT 335 N.D. BC040261.1 2432 3′UTR 322254CCCTTAGTGATCCCACCTTA 336 N.D. BC040261.1 2453 3′UTR 322255CTGCCCCATCCCTTAGTGAT 337 N.D. BC040261.1 2462 3′UTR 322256TTTATGTTTGGGCAGAGACT 338 N.D. BC040261.1 2480 3′UTR 322257CATGGCAGTCTATAACCCTT 339 N.D. BC040261.1 2556 3′UTR 322258TAGCATGGCAGTCTATAACC 340 N.D. BC040261.1 2559 3′UTR 322259TCTAGCATGGCAGTCTATAA 341 N.D. BC040261.1 2561 3′UTR 322260TTGTCTAGCATGGCAGTCTA 342 N.D. BC040261.1 2564 3′UTR 322261AAGCTTGTCTAGCATGGCAG 343 N.D. BC040261.1 2568 3′UTR 322262ACATGGACAAGCTTGTCTAG 344 N.D. BC040261.1 2576 3′UTR 322263TTACATGGACAAGCTTGTCT 345 N.D. BC040261.1 2578 3′UTR 322264GAATATTACATGGACAAGCT 346 N.D. BC040261.1 2583 3′UTR 322265AACTAGCCAGGTGCTAGGAG 347 N.D. BC040261.1 2636 3′UTR 322266AATTATTACTCACCACTGGG 348 N.D. NT_005543.12 1124 genomic 322267TAATATTTAGGGAAGCATGA 349 N.D. NT_005543.12 13890 genomic 322268GGACCCTGGGCCAGTTATTG 350 N.D. NT_005543.12 22504 genomic 322269CAAACATACCTGTCACAAAT 351 N.D. NT_005543.12 23662 genomic 322270GTGATATCAATTGATGGCAT 352 N.D. NT_005543.12 29265 genomic 322271TGCTACATCTACTCAGTGTC 353 N.D. NT_005543.12 31796 genomic 322272TGGAAACTCTTGCCTTCGGA 354 N.D. NT_005543.12 32971 genomic 322273CCATCCACATTGTAGCATGT 355 N.D. NT_005543.12 34646 genomic 322274TCAGGATGGTATGGCCATAC 356 N.D. NT_005543.12 36251 genomic 322275TCCCATAGTGCTAGAGTCGA 357 N.D. NT_005543.12 37218 genomic 322276AGGTTCTTACCAGAGAGCAG 358 N.D. NT_005543.12 37268 genomic 322277CAGAGGAGCAGCACCTAAAA 359 N.D. NT_005543.12 49133 genomic 322278GACCACATACCAAGCACTGA 360 N.D. NT_005543.12 49465 genomic 322279ATCTTTCAGAAACCCAAGCA 361 N.D. NT_005543.12 51347 genomic 322280GAGTCACCAAAGATTTACAA 362 N.D. NT_005543.12 51542 genomic 322281CTGAAGTTAGCTGAAAGCAG 363 N.D. NT_005543.12 51815 genomic 322282ACAGCTTTACCTATAGAGAA 364 N.D. NT_005543.12 52118 genonic 322283TCCTCAAGCTCTACAAATGA 365 N.D. NT_005543.12 54882 genomic 322284GACTCACTCACCACATTTAT 366 N.D. NT_005543.12 55027 genomic 322285AGTGATAGCAAGGCTTCTCT 367 N.D. NT_005543.12 56816 genomic 322286CTTGGAGAGAATGGTTATCT 368 N.D. NT_005543.12 61044 genomic 322287GAAGATGTTGATGCCTAAAT 369 N.D. NT_005543.12 63271 genomic 322288GTGTTGGTTCCTGAAAGACA 370 N.D. NT_005543.12 63665 genomic 322289CAGGATTTACCTTTTCTTGG 371 N.D. NT_005543.12 63711 genomic 322290AGGGCAGAATAGAGGTTGCC 372 N.D. NT_005543.12 64973 Genomic 322291TTTTTCTCTGGAGAAATAGA 373 N.D. NT_005543.12 65052 genomic 323624GTTACTCAGTCCCATAGTGC 374 59 U04343.1 143 START 323625CAAAGAGAATGTTACTCAGT 375 21 U04343.1 153 Coding 323626CCATCACAAAGAGAATGTTA 376 32 U04343.1 159 Coding 323627GGAAGGCCATCACAAAGAGA 377 54 U04343.1 165 Coding 323628GAGCAGGAAGGCCATCACAA 378 44 U04343.1 170 Coding 323629CCAGAGAGCAGGAAGGCCAT 379 36 U04343.1 175 Coding 323630AAATAAGCTTGAATCTTCAG 380 22 U04343.1 205 Coding 323631AGTCTCATTGAAATAAGCTT 381 56 U04343.1 215 Coding 323632AGGTCTGCAGTCTCATTGAA 382 41 U04343.1 223 Coding 323633CTACTAGCTCACTCAGGCTT 383 50 U04343.1 273 Coding 323634AAATACTACTAGCTCACTCA 384 30 U04343.1 278 Coding 323635CTGCCAAAATACTACTAGCT 385 24 U04343.1 284 Coding 323636TTCAGAACCAAGTTTTCCTG 386 23 U04343.1 307 Coding 323637CCTCATTCAGAACCAAGTTT 387 19 U04343.1 312 Coding 323638GTATACCTCATTCAGAACCA 388 20 U04343.1 317 Coding 323639GCCTAAGTATACCTCATTCA 389 55 U04343.1 323 Coding 323640CTCTTTGCCTAAGTATACCT 390 28 U04343.1 329 Coding 323641CCCATATACTTGGAATGAAC 391 88 U04343.1 361 Coding 323642CTTGTGCGGCCCATATACTT 392 27 U04343.1 370 Coding 323643ATCAAAACTTGTGCGGCCCA 393 80 U04343.1 377 Coding 323644CCCTTGTCCTTGATCTGAAG 394 71 U04343.1 427 Coding 323645ACAAGCCCTTGTCCTTGATC 395 56 U04343.1 432 Coding 323646TTGATACAAGCCCTTGTCCT 396 33 U04343.1 437 Coding 323647ATACATTGATACAAGCCCTT 397 41 U04343.1 442 Coding 323648TGGATGATACATTGATACAA 398 31 U04343.1 448 Coding 323649GAATTCATCTGGTGGATGCG 399 81 U04343.1 493 Coding 323650GTTCAGAATTCATCTGGTGG 400 92 U04343.1 498 Coding 323651TGACAGTTCAGAATTCATCT 401 64 U04343.1 503 Coding 323652AGCACTGACAGTTCAGAATT 402 87 U04343.1 508 Coding 323653TAGCAAGCACTGACAGTTCA 403 96 U04343.1 513 Coding 323654TGAAGTTAGCAAGCACTGAC 404 87 U04343.1 519 Coding 323655TTGACTGAAGTTAGCAAGCA 405 65 U04343.1 524 Coding 323656CTATTTCAGGTTGACTGAAG 406 76 U04343.1 534 Coding 323657TCTGTTATATTAGAAATTGG 407 43 U04343.1 556 Coding 323658GCAGGTCAAATTTATGTACA 408 36 U04343.1 581 Coding 323659GTATAGATGAGCAGGTCAAA 409 56 U04343.1 591 Coding 323660GGGTAACCGTGTATAGATGA 410 71 U04343.1 601 Coding 323661AGGTTCTGGGTAACCGTGTA 411 68 U04343.1 608 Coding 323662TAGCAAAACACTCATCTTCT 412 22 U04343.1 629 Coding 323663GTTCTTAGCAAAACACTCAT 413 23 U04343.1 634 Coding 323664ATTCTTGGTTCTTAGCAAAA 414 35 U04343.1 641 Coding 323665GATAGTTGAATTCTTGGTTC 415 43 U04343.1 650 Coding 323666ACCATCATACTCGATAGTTG 416 71 U04343.1 662 Coding 323667ATCTTGAGATTTCTGCATAA 417 52 U04343.1 683 Coding 323668ACATTATCTTGAGATTTCTG 418 39 U04343.1 688 Coding 323669CGTACAGTTCTGTGACATTA 419 68 U04343.1 702 Coding 323670AGACAAGCTGATGGAAACGT 420 19 U04343.1 722 Coding 323671GAAACAGACAAGCTGATGGA 421 26 U04343.1 727 Coding 323672GGAATGAAACAGACAAGCTG 422 33 U04343.1 732 Coding 323673CATCAGGGAATGAAACAGAC 423 38 U04343.1 738 Coding 323674CGTAACATCAGGGAATGAAA 424 47 U04343.1 743 Coding 323675AGCTCTATAGAGAAAGGTGA 425 77 U04343.1 817 Coding 323676CCTCAAGCTCTATAGAGAAA 426 24 U04343.1 822 Coding 323677GGAGGCTGAGGGTCCTCAAG 427 55 U04343.1 835 Coding 323678AGTACAGCTGTAATCCAAGG 428 23 U04343.1 868 Coding 323679TTGGAAGTACAGCTGTAATC 429 60 U04343.1 873 Coding 323680ATAATAACTGTTGGAAGTAC 430 51 U04343.1 883 Coding 323681CATCACACATATAATAACTG 431 8 U04343.1 893 Coding 323682TCCATTTCCATAGAATTAGA 432 35 U04343.1 921 Coding 323683TCTTCTTCCATTTCCATAGA 433 16 U04343.1 927 Coding 323684ATTTATAAGAGTTGCGAGGC 434 32 U04343.1 954 Coding 323685TTGGTTCCACATTTATAAGA 435 18 U04343.1 964 Coding 323686CTCTCCATTGTGTTGGTTCC 436 53 U04343.1 976 Coding 323687CTTCCCTCTCCATTGTGTTG 437 19 U04343.1 981 Coding 323688TGGTCTGTTCACTCTCTTCC 438 58 U04343.1 996 Coding 323689TTCATCAGATCTTTCAGGTA 439 43 U04343.1 1037 Coding 323690ATCACTTTTGTCGCATGAAG 440 82 U04343.1 1088 Coding 323691GCTTTACTCTTTAATTAAAA 441 40 U04343.1 1114 STOP 323692GTATGGGCTTTACTCTTTAA 442 57 U04343.1 1120 3′UTR 323693ATACTTGTATGGGCTTTACT 443 62 U04343.1 1126 3′UTR 323694AATGAATACTTGTATGGGCT 444 71 U04343.1 1131 3′UTR

1. A compound comprising a modified oligonucleotide consisting of 12 to50 linked nucleosides and having a nucleobase sequence comprising aportion of at least 8 contiguous nucleobases complementary to an equallength portion of nucleobases 493 to 532 of SEQ ID NO: 295, wherein thenucleobase sequence of the modified oligonucleotide is at least 90%complementary to SEQ ID NO: 295 as measured over the entirety of saidmodified oligonucleotide.
 2. The compound of claim 1, wherein themodified oligonucleotide is 100% complementary to an equal lengthportion of nucleobases 493 to 532 of SEQ ID NO: 295 as measured over theentirety of said modified oligonucleotide.
 3. The compound of claim 1,wherein the modified oligonucleotide is at least 95% complementary toSEQ ID NO: 295 as measured over the entirety of said modifiedoligonucleotide.
 4. The compound of claim 3, wherein the modifiedoligonucleotide is 100% complementary to SEQ ID NO: 295 as measured overthe entirety of said modified oligonucleotide.
 5. The compound of claim3, wherein the modified olignucleotide hybridizes exclusively withinnucleobases 493 to 532 of SEQ ID NO:
 295. 6. The compound of claim 4,wherein the modified olignucleotide hybridizes exclusively withinnucleobases 493 to 532 of SEQ ID NO:
 295. 7. The compound of claim 1,consisting of a single-stranded modified oligonucleotide.
 8. Thecompound of claim 7, comprising at least one modified internucleosidelinkage.
 9. The compound of claim 8, wherein each modifiedinternucleoside linkage is a phosphorothioate internucleoside linkage.10. The compound of claim 7, wherein at least one nucleoside comprises amodified sugar.
 11. The compound of claim 10, wherein the at least onemodified sugar is a bicyclic sugar.
 12. The compound of claim 10,wherein the at least one modified sugar comprises a 2′-O-methoxyethyl ora 4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1 or
 2. 13. The compound ofclaim 7, wherein at least one nucleoside comprises a modifiednucleobase.
 14. The compound of claim 13, wherein the modifiednucleobase is a 5-methylcytosine.
 15. The compound of claim 1, whereinthe modified oligonucleotide comprises: a gap segment consisting oflinked deoxynucleosides; a 5′ wing segment consisting of linkednucleosides; a 3′ wing segment consisting of linked nucleosides; whereinthe gap segment is positioned between the 5′ wing segment and the 3′wing segment and wherein each nucleoside of each wing segment comprisesa modified sugar.
 16. The compound of claim 15, wherein the modifiedoligonucleotide comprises: a gap segment consisting of ten linkednucleosides; a 5′ wing segment consisting of five linked nucleosides; a3′ wing segment consisting of five linked nucleosides; wherein the gapsegment is positioned between the 5′ wing segment and the 3′ wingsegment, wherein each nucleoside of each wing segment comprises a2′-O-methoxyethyl sugar; and wherein each internucleoside linkage ofsaid modified oligonucleotide is a phosphorothioate linkage.
 17. Thecompound of claim 7, wherein the modified oligonucleotide consists of 20linked nucleosides.
 18. A composition comprising the compound of claim1, or a salt thereof, and a pharmaceutically acceptable carrier ordiluent.
 19. The composition of claim 18, wherein the modifiedoligonucleotide consists of 20 linked nucleosides.
 20. The compositionof claim 18, wherein the modified oligonucleotide is 100% complementaryto an equal length portion of nucleobases 493 to 532 of SEQ ID NO: 295as measured over the entirety of said modified oligonucleotide.
 21. Amethod comprising administering to an individual a compound of claim 1.22. The method of claim 21, wherein the individual is a human.
 23. Themethod of claim 22, wherein administering the compound to the humantreats a disease or condition selected from the group consisting ofrheumatoid arthritis, multiple sclerosis, psoriasis, asthma, airwayhyperresponsiveness, and pulmonary inflammation.
 24. The method of claim21, comprising co-administering the compound and any of the groupconsisting of a steroid, montelukast sodium, albuterol, beclomethasonedipropionate, triamcinolone acetonide, ipratropium bromide, flunisolide,and fluticasone propionate.
 25. The method of claim 21, wherein theadministering is any of the group consisting of topical, pulmonary,inhalation, insufflation, intratracheal, intranasal, epidermal,transdermal, oral, parenteral, intravenous, intraarterial, subcutaneous,intraperitoneal, intramuscular, intracranial, intrathecal, andintraventricular administration.
 26. The method of claim 21, wherein themodified oligonucleotide is 100% complementary to an equal lengthportion of nucleobases 493 to 532 of SEQ ID NO: 295 as measured over theentirety of said modified oligonucleotide.